Postpartum cells derived from placental tissue, and methods of making and using the same

ABSTRACT

Cells derived from postpartum placenta and methods for their isolation are provided by the invention. The invention further provides cultures and compositions of the placenta-derived cells. The placenta-derived cells of the invention have a plethora of uses, including but not limited to research, diagnostic, and therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/877,446, filed Jun. 25, 2004 (now allowed), which claims benefit ofU.S. Provisional Application Ser. No. 60/483,264, filed Jun. 27, 2003,the entire contents of which are incorporated by reference herein. Thisapplication is also related to the following commonly-owned applicationsU.S. application Ser. No. 10/877,012, filed Jun. 25, 2004 (now U.S. Pat.No. 7,510,873, issued Mar. 31, 2009), U.S. application Ser. No.10/877,269, filed Jun. 25, 2004 (now U.S. Pat. No. 7,524,489, issuedApr. 28, 2009), U.S. application Ser. No. 10/877,445, filed Jun. 25,2004 (now U.S. Pat. No. 8,703,121, issued Apr. 22, 2014), U.S.application Ser. No. 10/877,541, filed Jun. 25, 2004 (now U.S. Pat. No.7,413,734, issued Aug. 19, 2008), U.S. application Ser. No. 10/877,009,filed Jun. 25, 2004 (now U.S. Pat. No. 7,560,276, issued Jul. 14, 2009),U.S. application Ser. No. 10/877,998, filed Jun. 25, 2004, and U.S.Provisional Application No. 60/555,908, filed Mar. 24, 2004, the entirecontents of each of which are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the field of mammalian cell biology and cellculture. In particular, the invention relates to cultured cells derivedfrom postpartum placental tissue having the potential to differentiateinto multiple lineages, and methods of preparation and use of thoseplacenta-derived cells.

BACKGROUND OF THE INVENTION

Organ and tissue generation from cells provides promising treatments fora number of pathologies, thereby making stem cells a central focus ofresearch in many fields. Human stem cells are capable of generating avariety of mature human cell lineages. Transplantation of such cells hasprovided a clinical tool for reconstituting a target tissue, therebyrestoring physiologic and anatomic functionality. The application ofstem cell technology is wide-ranging, including tissue engineering, genetherapy delivery, and cell therapeutics for disorders includingmalignancies, inborn errors of metabolism, hemoglobinopathies, andimmunodeficiences.

An obstacle to realization of the therapeutic potential of stem celltechnology has been difficulty in obtaining sufficient numbers of humanstem cells. One source of stem cells is embryonic or fetal tissue.Embryonic stem and progenitor cells have been isolated from a number ofmammalian species, including humans. The derivation of stem cells fromembryonic or fetal sources, however, has raised many ethical and moralissues.

Stem cells also have been isolated from adult tissues. Methods forisolation of stem cells from adult sources often yield only limitedquantities of cells and/or cells having limited ability todifferentiate.

Postpartum tissues have generated interest as an alternative source forhuman stem cells. For example, methods for recovery of stem cells byperfusion of the placenta or collection from umbilical cord blood havebeen described. A limitation of stem cell procurement from these methodshas been an inadequate volume of cord blood or quantity of cellsobtained.

Thus, alternative sources of adequate supplies of cells having theability to differentiate into an array of cell lineages forcryopreservation and/or use in clinical applications remain in greatdemand. Such cells may be used in drug screening assays, forcryopreservation and/or banking, and for diagnostic and therapeuticapplications.

SUMMARY OF THE INVENTION

The present invention relates to cells derived from postpartum placenta.The cells of the invention may be characterized by any one or more ofcharacteristics including the presence or absence of cell surfacemarkers, methods of extraction from placental tissue, gene expressionprofiles, protein production profiles, secretion of factors, growthcharacteristics, or any combination of such characteristics.

The invention encompasses cells derived from human postpartum placentaltissue substantially free of blood. In some embodiments, the cell iscapable of self-renewal and expansion in culture. In some aspects of theinvention, the cell has the potential to differentiate into cells ofanother phenotype. In some embodiments, the placenta-derived cellrequires L-valine for growth. The placenta-derived cells of theinvention are capable of growth in about 5% to about 20% oxygen. In someembodiments of the invention, the placenta-derived cell exhibits atleast one of the following characteristics:

(a) production of at least one of tissue factor, vimentin, granulocytechemotactic protein-2 (GCP-2), and alpha-smooth muscle actin;

(b) lack of production of at least one of GRO-alpha and oxidized lowdensity lipoprotein receptor, as detected by flow cytometry;

(c) production of at least one of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, PD-L2 and HLA-A,B,C;

(d) lack of production of at least one of CD31, CD34, CD45, CD80, CD86,CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DP, DQ, DR, as detected byflow cytometry;

(e) expression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an iliac crest bone marrow cell, is increasedfor at least one of C-type lectin superfamily member A2, Wilms tumor 1,aldehyde dehydrogenase 1 family member A2, renin, oxidized low densitylipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671,hypothetical protein DKFZp564F013, downregulated in ovarian cancer 1,and clone DKFZp547K1113;

(f) expression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an iliac crest bone marrow cell, is reducedfor at least one of: short stature homeobox 2; heat shock 27 kDa protein2; chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);elastin; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeobox 2; sine oculis homeobox homolog 1; crystallin, alpha B;dishevelled associated activator of morphogenesis 2; DKFZP586B2420protein; similar to neuralin 1; tetranectin; src homology three (SH3)and cysteine rich domain; B-cell translocation gene 1,anti-proliferative; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; hypothetical protein FLJ23191; interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog7; hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C;iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; cDNA FLJ12280 fis, clone MAMMA1001744; cytokinereceptor-like factor 1; potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 4; integrin, alpha 7;DKFZP586L151 protein; transcriptional co-activator with PDZ-bindingmotif (TAZ); sine oculis homeobox homolog 2; KIAA1034 protein; earlygrowth response 3; distal-less homeobox 5; hypothetical proteinFLJ20373; aldo-keto reductase family 1, member C3 (3-alphahydroxysteroid dehydrogenase, type II); biglycan; fibronectin 1;proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains);cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; cDNA DKFZp564B222 (fromclone DKFZp564B222); vesicle-associated membrane protein 5;EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; and insulin-like growthfactor binding protein 2, 36 kDa;

(g) secretion of at least one of monocyte chemotactic protein 1 (MCP-1),interleukin-6 (IL-6), stromal-derived factor 1 alpha (SDF-1alpha),interleukin 8 (IL8), granulocyte chemotactic protein-2 (GCP-2),hepatocyte growth factor (HGF), keratinocyte growth factor (KGF),heparin-binding epidermal growth factor (HB-EGF), brain-derivedneurotrophic factor (BDNF), tissue inhibitor of matrix metalloproteinase1 (TIMP1), thrombopoietin (TPO), macrophage inflammatory protein 1alpha(MIP1a), Rantes (regulated on activation, normal T cell expressed andsecreted), thymus and activation-regulated chemokine (TARC), andEotaxin;

(h) lack of secretion of at least one of fibroblast growth factor (FGF),vascular endothelial growth factor (VEGF), angiopoietin 2 (ANG2),platelet derived growth factor (PDGF-bb), transforming growth factorbeta2 (TGFbeta2), macrophage inflammatory protein 1beta (MIP1b), 1309,and macrophage-derived chemokine (MDC), as detected by ELISA; and

(i) the ability to undergo at least 40 population doublings in culture.

In specific embodiments, the cell has all identifying features of anyone of: cell type PLA 071003 (P8) (ATCC Accession No. PTA-6074); celltype PLA 071003 (P11) (ATCC Accession No. PTA-6075); and cell type PLA071003 (P16) (ATCC Accession No. PTA-6079). The placenta-derived cellsof the invention are preferably human cells. The cells of the inventionmay be of neonatal lineage, maternal lineage, or a combination thereof.

The invention also provides placenta-derived cells isolated from apost-partum placenta or fragment thereof by enzymatic dissociation witha matrix metalloprotease (MMP); a matrix metalloprotease and a neutralprotease; a matrix metalloprotease and a mucolytic enzyme that digestshyaluronic acid; or a matrix metalloprotease, a neutral protease, and amucolytic enzyme that digests hyaluronic acid. Preferable matrixmetalloproteases include collagenase. The neutral protease is preferablythermolysin or dispase, and most preferably is dispase. The mucolyticenzyme that digests hyaluronic acid preferably is hyaluronidase. TheLIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.) Blendzyme(Roche) series of enzyme combinations are very useful and may be used inthe instant methods. Other sources of enzymes are known, and the skilledartisan may also obtain such enzymes directly from their naturalsources. The skilled artisan is also well-equipped to assess new, oradditional enzymes or enzyme combinations for their utility in isolatingthe cells of the invention. Preferred enzyme treatments are 0.5, 1, 1.5,or 2 hours long or longer. In more preferred embodiments, the tissue isincubated at 37° C. during the enzyme treatment of the disintegrationstep.

In some embodiments of the invention, the placental tissue is separatedinto fractions prior to cell extraction, such that the cell ispredominantly of neonatal or maternal derivation. In some aspects of theinvention, placental tissue is mechanically dissociated prior to thestep of enzymatic dissociation. In some embodiments, the method ofisolation of the cells of the invention further involves growing thecells in culture medium. The culture medium preferably is RPMI1640,Ham's F10 medium, Ham's F12 medium, Mesenchymal Stem Cell Growth Medium,Iscove's modified Dulbecco's medium, Dulbecco's modified Eagle's Medium(DMEM), advanced DMEM (Gibco), DMEM/MCDB201 (Sigma), CELL-GRO FREE,DMEM/F12, or Eagle's basal medium. In some aspects of the invention, theculture medium is supplemented with about 2% to about 15% (v/v) serum,beta-mercaptoethanol, glucose, and/or an antibiotic agent and anantimycotic agent. The culture medium preferably is Growth mediumcomprising DMEM, glucose, beta-mercaptoethanol, serum, and an antibioticagent. The culture medium may contain at least one of fibroblast growthfactor, platelet-derived growth factor, vascular endothelial growthfactor, epidermal growth factor, and leukemia inhibitory factor. Thecells of the invention may be grown on an uncoated or coated surface.Surfaces for growth of the cells may be coated for example with gelatin,collagen (e.g., native or denatured), fibronectin, laminin, ornithine,vitronectin, or extracellular membrane protein (e.g., MATRIGEL (BDDiscovery Labware, Bedford, Mass.)).

The invention includes within its scope placenta-derived cellscharacterized by growth characteristics, such as but not limited to,cells that yield greater than about 10¹⁷ cells in about 60 days uponinitial seeding at about 1,000 to about 5,000 cells/cm². In someembodiments, the placenta-derived cells of the invention have theability to undergo at least 40 population doublings in about 80 days inculture.

The placenta-derived cells of the invention may be utilized from thefirst subculture (passage 0) to senescence. The preferable number ofpassages is that which yields a cell number sufficient for a givenapplication. In certain embodiments, the cells are passaged 2 to 25times, preferably 4 to 20 times, more preferably 8 to 15 times, morepreferably 10 or 11 times, and most preferably 11 times.

Methods for inducing differentiation of placenta-derived cells of theinvention also are contemplated. In some embodiments of the invention,placenta-derived cells are induced to a mesodermal, ectodermal, orendodermal lineage. For example, the cells may be induced todifferentiate to an adipogenic, a chondrogenic, an osteogenic, aneurogenic, an oculogenic, a pancreagenic, a cardiomyogenic, or ahepatogenic lineage. Methods of inducing differentiation of the cells ofthe invention preferably involve contacting or exposing the cells to oneor more differentiation-inducing agents. In some embodiments, suchcontact or exposure occurs in culture. The invention includes the cellsso induced.

Cells of the invention may be genetically engineered to express a geneof interest or to produce a protein of interest such as but not limitedto a therapeutic protein. For example, PDCs may be geneticallyengineered to express an antiinflammatory compound or an anti-apoptoticagent.

Methods of the invention further include methods for producing apopulation of placenta-derived cells by expanding a cell or cells of theinvention in culture. The PDCs may be differentiation-induced orundifferentiated. In some embodiments, a population of placenta-derivedcells is mixed with another population of cells. In some embodiments,the cell population is heterogeneous. A heterogeneous cell population ofthe invention may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% undifferentiated or differentiation-inducedPDCs of the invention. The heterogeneous cell populations of theinvention may further comprise stem cells or cells of a mesodermal,endodermal, or ectodermal lineage. Cell populations of the invention maybe homogeneous. Homogeneous populations of placenta-derived cells may beof neonatal or maternal lineage. Homogeneity of a cell population may beachieved by any method known in the art, for example, by cell sorting(e.g., flow cytometry) or by clonal expansion.

Some embodiments of the invention provide methods of manufacturing atissue matrix for implantation into a patient by seeding one or moreplacenta-derived cells of the invention onto or into a tissue matrix forimplantation into a patient. The PDCs may be differentiated orundifferentiated. The matrix may contain one or more factors includingdrugs, anti-apoptotic agents (e.g., erythropoietin (EPO), EPOmimetibody, thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II,hepatocyte growth factor, caspase inhibitors), anti-inflammatorycompounds (e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors,statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE,SIROLIMUS, and non-steroidal anti-inflammatory drugs (NSAIDS) (such asTEPDXALIN, TOLMETIN, and SUPROFEN)) as well as local anesthetics, andgrowth factors. In some aspects of the invention, the matrix comprisesdecellularized tissue, such as extracellular matrix or cell lysates ofthe PDCs. In some embodiments, the matrix is biodegradable. In someaspects of the invention, the matrix comprises natural or syntheticpolymers. Matrices of the invention include biocompatible scaffolds,lattices, self-assembling structures and the like, whether biodegradableor not, liquid or solid. Such matrices are known in the arts ofcell-based therapy, surgical repair, tissue engineering, and woundhealing. Preferably the matrices are pretreated (e.g., seeded,inoculated, contacted with) with the cells, extracellular matrix,conditioned medium, cell lysate, or combination thereof, of theinvention. More preferably the matrices are populated with cells inclose association to the matrix or its spaces. In some aspects of theinvention, the cells adhere to the matrix. In some embodiments, thecells are contained within or bridge interstitial spaces of the matrix.Most preferred are those seeded matrices wherein the cells are in closeassociation with the matrix and which, when used therapeutically, induceor support ingrowth of the patient's cells and/or proper angiogenesis.The seeded matrices can be introduced into a patient's body in any wayknown in the art, including but not limited to implantation, injection,surgical attachment, transplantation with other tissue, injection, andthe like. Examples of scaffolds which may be used in the presentinvention include nonwoven mats, porous foams, or self-assemblingpeptides. Nonwoven mats may, for example, be formed using fiberscomprised of a synthetic absorbable copolymer of glycolic and lacticacids (PGA/PLA) sold under the tradename VICRYL (Ethicon, Inc.Somerville, N.J.). Foams composed of, for example,poly(epsilon-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer,formed by the processes such as freeze-drying, or lyophilized, asdiscussed in U.S. Pat. No. 6,355,699, also are possible scaffolds.Hydrogels such as self-assembling peptides (e.g., RAD16) may also beused. These materials are frequently used as supports for growth oftissue. The matrices of the invention may be configured to the shapeand/or size of a tissue or organ in vivo. The scaffolds of the inventionmay be flat or tubular or may comprise sections thereof. The scaffoldsof the invention may be multilayered. Organs and tissues comprisingPDCs, their extracellular matrix, or cell lysate also are provided.

Also encompassed within the scope of the invention are extracellularmatrices of PDCs, cell fractions (e.g., soluble cell fractions) of PDCs,and PDC-conditioned medium.

In some embodiments the invention provides compositions of PDCs and oneor more bioactive factors, for example, but not limited to growthfactors, anti-apoptotic agents, anti-inflammatory agents, and/ordifferentiation inducing factors.

The cells, matrices, tissues, and compositions of the invention may becryopreserved. Cryopreserved cells and compositions of the invention maybe banked or stored. Methods for cryopreserving and/or storingpostpartum-derived cells of the invention also are contemplated.

Compositions of PDCs and related products, including for examplepharmaceutical compositions, are included within the scope of theinvention. Compositions of PDCs may include one or more of adifferentiation-inducing factor, a cell survival factor such as caspaseinhibitor, an anti-inflammatory agent such as p38 kinase inhibitor,growth factors, such as PDGF-bb, EGF, bFGF, LIF, IGF-1, or VEGF, or anangiogenic factor such as VEGF or bFGF. Pharmaceutical compositions ofthe placenta-derived cells, extracellular matrix produced thereby, celllysates thereof, and PDC-conditioned medium are included within thescope of the invention. The pharmaceutical compositions preferablyinclude a pharmaceutically acceptable carrier or excipient.

In some embodiments, methods of transplanting placenta-derived cells ormatrices and methods of regenerating a tissue or organ in a patient inneed thereof by transplanting cells or matrices of the invention into apatient are provided.

Further provided by the invention are methods for treating a disease orinjury in a patient by administering one or more placenta-derived cells,PDC populations, matrices, cell lysates, conditioned medium, orcompositions of the invention.

The invention also encompasses cell cultures of the placenta-derivedcells of the invention. The cultures of the invention preferably arecapable of at least 40 population doublings upon initial seeding.

The cell and compositions of the invention may be used, for example, inthe treatment of conditions or repair of tissue. In some embodiments ofthe invention, the condition to be treated is a condition of soft tissue(e.g., skin, muscle, vasculature, tendons, ligaments, bladder, fascia,pelvic floor), bone, pancreas, kidney, liver, nervous system, eye,heart, or cartilage.

Methods of the invention further include methods for producing apopulation of placenta-derived cells by expanding a cell of theinvention in culture.

Other features and advantages of the invention will be apparent from thedetailed description and examples that follow.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

Various terms used throughout the specification and claims are definedas set forth below.

Stem cells are undifferentiated cells defined by their ability at thesingle cell level to both self-renew and differentiate to produceprogeny cells, including self-renewing progenitors, non-renewingprogenitors and terminally differentiated cells. Stem cells are alsocharacterized by their ability to differentiate in vitro into functionalcells of various cell lineages from multiple germ layers (endoderm,mesoderm and ectoderm), as well as to give rise to tissues of multiplegerm layers following transplantation and to contribute substantially tomost, if not all, tissues following injection into blastocysts.

Stem cells are classified by their developmental potential as: (1)totipotent—able to give rise to all embryonic and extraembryonic celltypes; (2) pluripotent—able to give rise to all embryonic cell types;(3) multipotent—able to give rise to a subset of cell lineages, but allwithin a particular tissue, organ, or physiological system (for example,hematopoietic stem cells (HSC) can produce progeny that include HSC(self-renewal), blood cell-restricted oligopotent progenitors, and allcell types and elements (e.g., platelets) that are normal components ofthe blood); (4) oligopotent—able to give rise to a more restrictedsubset of cell lineages than multipotent stem cells; and (5)unipotent—able to give rise to a single cell lineage (e.g.,spermatogenic stem cells).

Stem cells are also categorized on the basis of the source from whichthey may be obtained. An adult stem cell is generally a multipotentundifferentiated cell found in tissue comprising multiple differentiatedcell types. The adult stem cell can renew itself and, under normalcircumstances, differentiate to yield the specialized cell types of thetissue from which it originated, and possibly other tissue types. Anembryonic stem cell is a pluripotent cell from the inner cell mass of ablastocyst-stage embryo. A fetal stem cell is one that originates fromfetal tissues or membranes. A postpartum stem cell is a multipotent orpluripotent cell that originates substantially from extraembryonictissue available after birth, namely, the placenta and the umbilicalcord. These cells have been found to possess features characteristic ofpluripotent stem cells, including rapid proliferation and the potentialfor differentiation into many cell lineages. Postpartum stem cells maybe blood-derived (e.g., as are those obtained from umbilical cord blood)or non-blood-derived (e.g., as obtained from the non-blood tissues ofthe umbilical cord and placenta).

Embryonic tissue is typically defined as tissue originating from theembryo (which in humans refers to the period from fertilization to aboutsix weeks of development. Fetal tissue refers to tissue originating fromthe fetus, which in humans refers to the period from about six weeks ofdevelopment to parturition. Extraembryonic tissue is tissue associatedwith, but not originating from, the embryo or fetus. Extraembryonictissues include extraembryonic membranes (chorion, amnion, yolk sac andallantois), umbilical cord, and placenta (which itself forms from thechorion and the maternal decidua basalis).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cell,such as a nerve cell or a muscle cell, for example. A differentiated ordifferentiation-induced cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term committed,when applied to the process of differentiation, refers to a cell thathas proceeded in the differentiation pathway to a point where, undernormal circumstances, it will continue to differentiate into a specificcell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. De-differentiation refers to the processby which a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell. As used herein, the lineage of a celldefines the heredity of the cell, i.e., which cells it came from andwhat cells it can give rise to. The lineage of a cell places the cellwithin a hereditary scheme of development and differentiation. Alineage-specific marker refers to a characteristic specificallyassociated with the phenotype of cells of a lineage of interest and canbe used to assess the differentiation of an uncommitted cell to thelineage of interest.

In a broad sense, a progenitor cell is a cell that has the capacity tocreate progeny that are more differentiated than itself and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in greater detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a non-renewing progenitor cell or as anintermediate progenitor or precursor cell.

As used herein, the phrase differentiates into a mesodermal, ectodermalor endodermal lineage refers to a cell that becomes committed to aspecific mesodermal, ectodermal or endodermal lineage, respectively.Examples of cells that differentiate into a mesodermal lineage or giverise to specific mesodermal cells include, but are not limited to, cellsthat are adipogenic, chondrogenic, cardiogenic, dermatogenic,hematopoietic, hemangiogenic, myogenic, nephrogenic, urogenitogenic,osteogenic, pericardiogenic, or stromal. Examples of cells thatdifferentiate into ectodermal lineage include, but are not limited toepidermal cells, neurogenic cells, and neurogliagenic cells. Examples ofcells that differentiate into endodermal lineage include, but are notlimited to pleurigenic cells, and hepatogenic cells, cell that give riseto the lining of the intestine, and cells that give rise to pancreogenicand splanchogenic cells.

The cells of the present invention are referred to as placenta-derivedcells (PDCs). They also may sometimes be referred to herein aspostpartum-derived cells or postpartum cells (PPDCs). In addition, thecells may be described as being stem or progenitor cells, the latterterm being used in the broad sense. The term derived is used to indicatethat the cells have been obtained from their biological source and grownor otherwise manipulated in vitro (e.g., cultured in a growth medium toexpand the population and/or to produce a cell line). The in vitromanipulations of placenta-derived cells and the unique features of theplacenta-derived cells of the present invention are described in detailbelow.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition (“in culture”). A primary cell culture is a cultureof cells, tissues or organs taken directly from organisms and before thefirst subculture. Cells are expanded in culture when they are placed ina growth medium under conditions that facilitate cell growth and/ordivision, resulting in a larger population of the cells. When cells areexpanded in culture, the rate of cell proliferation is sometimesmeasured by the amount of time needed for the cells to double in number.This is referred to as doubling time.

A cell line is a population of cells formed by one or moresubcultivations of a primary cell culture. Each round of subculturing isreferred to as a passage. When cells are subcultured, they are referredto as having been passaged. A specific population of cells, or a cellline, is sometimes referred to or characterized by the number of timesit has been passaged. For example, a cultured cell population that hasbeen passaged ten times may be referred to as a P10 culture. The primaryculture, i.e., the first culture following the isolation of cells fromtissue, is designated P0. Following the first subculture, the cells aredescribed as a secondary culture (P1 or passage 1). After the secondsubculture, the cells become a tertiary culture (P2 or passage 2), andso on. It will be understood by those of skill in the art that there maybe many population doublings during the period of passaging; thereforethe number of population doublings of a culture is greater than thepassage number. The expansion of cells (i.e., the number of populationdoublings) during the period between passaging depends on many factors,including but not limited to the seeding density, substrate, medium, andtime between passaging.

A conditioned medium is a medium in which a specific cell or populationof cells has been cultured, and then removed. While the cells arecultured in the medium, they secrete cellular factors that can providetrophic support to other cells. Such trophic factors include, but arenot limited to hormones, cytokines, extracellular matrix (ECM),proteins, vesicles, antibodies, and granules. The medium containing thecellular factors is the conditioned medium.

Generally, a trophic factor is defined as a substance that promotessurvival, growth, proliferation, maintenance, differentiation, and/ormaturation of a cell, or stimulates increased activity of a cell.

When referring to cultured vertebrate cells, the term senescence (alsoreplicative senescence or cellular senescence) refers to a propertyattributable to finite cell cultures; namely, their inability to growbeyond a finite number of population doublings (sometimes referred to asHayflick's limit). Although cellular senescence was first describedusing fibroblast-like cells, most normal human cell types that can begrown successfully in culture undergo cellular senescence. The in vitrolifespan of different cell types varies, but the maximum lifespan istypically fewer than 100 population doublings (this is the number ofdoublings for all the cells in the culture to become senescent and thusrender the culture unable to divide). Senescence does not depend onchronological time, but rather is measured by the number of celldivisions, or population doublings, the culture has undergone. Thus,cells made quiescent by removing essential growth factors are able toresume growth and division when the growth factors are re-introduced,and thereafter carry out the same number of doublings as equivalentcells grown continuously. Similarly, when cells are frozen in liquidnitrogen after various numbers of population doublings and then thawedand cultured, they undergo substantially the same number of doublings ascells maintained unfrozen in culture. Senescent cells are not dead ordying cells; they are actually resistant to programmed cell death(apoptosis), and have been maintained in their nondividing state for aslong as three years. These cells are very much alive and metabolicallyactive, but they do not divide. The nondividing state of senescent cellshas not yet been found to be reversible by any biological, chemical, orviral agent.

As used herein, the term Growth medium refers to a culture mediumsufficient for expansion of placenta-derived cells. The culture mediumof Growth medium preferably contains Dulbecco's Modified Essential Media(DMEM). More preferably, Growth medium contains glucose. Growth mediumpreferably contains DMEM-low glucose (DMEM-LG) (Invitrogen, Carlsbad,Calif.). Growth medium preferably contains about 15% (v/v) serum (e.g.,fetal bovine serum, defined bovine serum). Growth medium preferablycontains at least one antibiotic agent and/or antimycotic agent (e.g.,penicillin, streptomycin, amphotericin B, gentamicin, nystatin;preferably 50 units/milliliter penicillin G sodium and 50micrograms/milliliter streptomycin sulfate). Growth medium preferablycontains 2-mercaptoethanol (Sigma, St. Louis Mo.). Most preferably,Growth medium contains DMEM-low glucose, serum, 2-mercaptoethanol, andan antibiotic agent and antimycotic agent.

As used herein, standard growth conditions refers to standardatmospheric conditions comprising 5% CO₂ and a temperature in the rangeof 35° C. to 39° C., more preferably, 37° C., and a relative humidity ofabout 100%.

The term isolated refers to a cell, cellular component, or a moleculethat has been removed from its native environment. PDCs, for example,may be isolated in some embodiments of the invention.

The term about refers to an approximation of a stated value within arange of ±10%.

The term treating (or treatment of) a condition refers to amelioratingthe effects of, or delaying, halting or reversing the progress of, ordelaying or preventing the onset of, a condition such as but not limitedto a congenital anomaly, disease, or injury.

The term effective amount refers to a concentration of a reagent orpharmaceutical composition, such as a growth factor, differentiationagent, trophic factor, cell population or other agent, that is effectivefor producing an intended result, including cell growth and/ordifferentiation in vitro or in vivo, or treatment of a condition asdescribed herein. With respect to growth factors, an effective amountmay range from about 1 nanogram/milliliter to about 1microgram/milliliter. With respect to PDCs as administered to a patientin vivo, an effective amount may range from as few as several hundred orfewer to as many as several million or more. In specific embodiments, aneffective amount may range from 10³-10¹¹. It will be appreciated thatthe number of cells to be administered will vary depending on thespecifics of the disorder to be treated, including but not limited tosize or total volume/surface area to be treated, as well as proximity ofthe site of administration to the location of the region to be treated,among other factors familiar to the medicinal biologist.

The terms effective period (or time) and effective conditions refer to aperiod of time or other controllable conditions (e.g., temperature,humidity for in vitro methods), necessary or preferred for an agent orpharmaceutical composition to achieve its intended result.

The term patient or subject refers to animals, including mammals,preferably humans, who are treated with the pharmaceutical compositionsor in accordance with the methods described herein.

The term matrix as used herein refers to a support for the PPDCs of theinvention, for example, a scaffold (e.g., VICRYL, PCL/PGA, or RAD16) orsupporting medium (e.g., hydrogel, extracellular membrane protein (e.g.,MATRIGEL (BD Discovery Labware, Bedford, Mass.)).

The term pharmaceutically acceptable carrier (or medium), which may beused interchangeably with the term biologically compatible carrier ormedium, refers to reagents, cells, compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other complication commensurate with a reasonable benefit/risk ratio.As described in greater detail herein, pharmaceutically acceptablecarriers suitable for use in the present invention include liquids,semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds). Asused herein, the term biodegradable describes the ability of a materialto be broken down (e.g., degraded, eroded, dissolved) in vivo. The termincludes degradation in vivo with or without elimination (e.g., byresorption) from the body. The semi-solid and solid materials may bedesigned to resist degradation within the body (non-biodegradable) orthey may be designed to degrade within the body (biodegradable,bioerodable). A biodegradable material may further be bioresorbable orbioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids(water-soluble implants are one example), or degraded and ultimatelyeliminated from the body, either by conversion into other materials orbreakdown and elimination through natural pathways.

Several terms are used herein with respect to cell replacement therapy.The terms autologous transfer, autologous transplantation, autograft andthe like refer to treatments wherein the cell donor is also therecipient of the cell replacement therapy. The terms allogeneictransfer, allogeneic transplantation, allograft and the like refer totreatments wherein the cell donor is of the same species as therecipient of the cell replacement therapy, but is not the sameindividual. A cell transfer in which the donor's cells have beenhistocompatibly matched with a recipient is sometimes referred to as asyngeneic transfer. The terms xenogeneic transfer, xenogeneictransplantation, xenograft and the like refer to treatments wherein thecell donor is of a different species than the recipient of the cellreplacement therapy.

The following abbreviations are used herein:

ANG2 (or Ang2) for angiopoietin 2;

APC for antigen-presenting cells;

BDNF for brain-derived neurotrophic factor;

bFGF for basic fibroblast growth factor;

bid (BID) for “bis in die” (twice per day);

BSP for bone sialoprotein;

CK18 for cytokeratin 18;

CXC ligand 3 for chemokine receptor ligand 3;

DAPI for 4′-6-Diamidino-2-phenylindole-2HCl;

DMEM for Dulbecco's Minimal Essential Medium;

DMEM:lg (or DMEM:Lg, DMEM:LG) for DMEM with low glucose;

EDTA for ethylene diamine tetraacetic acid;

EGF (or E) for epidermal growth factor;

EPO for erythropoietin;

FACS for fluorescent activated cell sorting;

FBS for fetal bovine serum;

FGF (or F) for fibroblast growth factor;

GCP-2 for granulocyte chemotactic protein-2;

GDF-5 for growth and differentiation factor 5;

GFAP for glial fibrillary acidic protein;

HB-EGF for heparin-binding epidermal growth factor;

HCAEC for Human coronary artery endothelial cells;

HGF for hepatocyte growth factor;

hMSC for Human mesenchymal stem cells;

HNF-1alpha for hepatocyte-specific transcription factor;

HUVEC for Human umbilical vein endothelial cells;

1309 for a chemokine and the ligand for the CCR8 receptor and isresponsible for chemoattraction of TH2 type T-cells;

IGF for insulin-like growth factor;

IL-6 for interleukin-6;

IL-8 for interleukin 8;

K19 for keratin 19;

K8 for keratin 8;

KGF for keratinocyte growth factor;

MCP-1 for monocyte chemotactic protein 1;

MDC for macrophage-derived chemokine;

MIP1alpha for macrophage inflammatory protein 1alpha;

MIP1beta for macrophage inflammatory protein 1beta;

MMP for matrix metalloprotease (MMP);

MSC for mesenchymal stem cells;

NHDF for Normal Human Dermal Fibroblasts;

NPE for Neural Progenitor Expansion media;

OxLDLR for oxidized low density lipoprotein receptor;

PBMC for peripheral blood mononuclear cell;

PBS for phosphate buffered saline;

PDC for placenta-derived cell;

PDGFbb for platelet derived growth factor;

PDGFr-alpha for platelet derived growth factor receptor alpha;

PD-L2 for programmed-death ligand 2;

PE for phycoerythrin;

PO for “per os” (by mouth);

PPDC for postpartum-derived cell;

Rantes (or RANTES) for regulated on activation, normal T cell expressedand secreted;

rb for rabbit

rh for recombinant;

SC for subcutaneously;

SCID for severe combined immunodeficiency;

SDF-1alpha for stromal-derived factor 1alpha;

SHH for sonic hedgehog;

SMA for smooth muscle actin;

SOP for standard operating procedure;

TARC for thymus and activation-regulated chemokine;

TCP for tissue culture plastic;

TGFbeta2 for transforming growth factor beta2;

TGFbeta-3 for transforming growth factor beta-3;

TIMP1 for tissue inhibitor of matrix metalloproteinase 1;

TPO for thrombopoietin;

TuJ1 for BIII Tubulin;

UDC for umbilical cord-derived cell;

VEGF for vascular endothelial growth factor;

vWF for von Willebrand factor; and

alphaFP for alpha-fetoprotein.

DESCRIPTION

Various patents and other publications are cited herein and throughoutthe specification, each of which is incorporated by reference herein inits entirety.

In one aspect, the invention provides placenta-derived cells (PDCs)derived from placental tissue washed substantially free of blood. ThePDCs may be derived from placenta of a mammal including but not limitedto human. The placentas from which the cells are derived are post-partumplacentas. The cells are capable of self-renewal and expansion inculture. The placenta-derived cells have the potential to differentiateinto cells of other phenotypes. In preferred embodiments, the cells candifferentiate into a cell of ectodermal, mesodermal, or endodermalorigin. The invention provides, in one of its several aspects, cellsthat are isolated from placental tissues, as opposed to placental blood.

The cells have been characterized as to several of their cellular,genetic, immunological, and biochemical properties. For example, thecells have been characterized by their growth, by their cell surfacemarkers, by their gene expression, by their ability to produce certainbiochemical trophic factors, and by their immunological properties.

Derivation and Expansion of Placenta-Derived Cells (PDCs)

According to the methods described herein, a mammalian placenta isrecovered upon or shortly after termination of either a full-term orpre-term pregnancy, for example, after its expulsion after birth.Placental tissue can be obtained from any completed pregnancy, full-termor less than full-term, whether delivered vaginally, or through othermeans, for example, Cessarian section. The placenta may be transportedfrom the birth site to a laboratory in a sterile container such as aflask, beaker, culture dish, or bag. The container may have a solutionor medium, including but not limited to a salt solution, such as, forexample, Dulbecco's Modified Eagle's Medium (DMEM) or phosphate bufferedsaline (PBS), or any solution used for transportation of organs used fortransplantation, such as University of Wisconsin solution orperfluorochemical solution. One or more antibiotic and/or antimycoticagents, such as but not limited to penicillin, streptomycin,amphotericin B, gentamicin, and nystatin, may be added to the medium orbuffer. The placenta may be rinsed with an anticoagulant solution suchas heparin-containing solution. It is preferable to keep the tissue atabout 4-10° C. prior to extraction of PDCs. It is even more preferablethat the tissue not be frozen prior to extraction of PDCs.

Isolation of PDCs preferably occurs in an aseptic environment. Theumbilical cord is removed from the placenta by means known in the art.Placental tissue is washed substantially free of blood and debris priorto derivation of PDCs. For example, the placental tissue may be washedwith buffer solution, such as but not limited to phosphate bufferedsaline. The wash buffer also may comprise one or more antimycotic and/orantibiotic agents, such as but not limited to penicillin, streptomycin,amphotericin B, gentamicin, and nystatin.

In some aspects of the invention, the different cell types present inpostpartum tissue are fractionated into subpopulations from which thePDCs can be isolated. This may be accomplished using techniques for cellseparation including, but not limited to, enzymatic treatment todissociate postpartum tissue into its component cells, followed bycloning and selection of specific cell types, for example but notlimited to selection based on morphological and/or biochemical markers;selective growth of desired cells (positive selection), selectivedestruction of unwanted cells (negative selection); separation basedupon differential cell agglutinability in the mixed population as, forexample, with soybean agglutinin; freeze-thaw procedures; differentialadherence properties of the cells in the mixed population; filtration;conventional and zonal centrifugation; centrifugal elutriation(counter-streaming centrifugation); unit gravity separation;countercurrent distribution; electrophoresis; and flow cytometry, forexample, fluorescence activated cell sorting (FACS).

In a preferred embodiment, placental tissue comprising a whole placentaor a fragment or section thereof is disaggregated by mechanical force(mincing or shear forces), enzymatic digestion with single orcombinatorial proteolytic enzymes, such as a matrix metalloproteaseand/or neutral protease, for example, collagenase, trypsin, dispase,LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.), hyaluronidase,and/or pepsin, or a combination of mechanical and enzymatic methods. Forexample, the cellular component of the placental tissue may bedisaggregated by methods using collagenase-mediated dissociation.Collagenase may be type 1, 2, 3, or 4. Enzymatic digestion methodspreferably employ a combination of enzymes, such as a combination of amatrix metalloprotease and a neutral protease, for example, acombination of collagenase and dispase. More preferably, enzymaticdigestion of placental tissue uses a combination of a matrixmetalloprotease, a neutral protease, and a mucolytic enzyme fordigestion of hyaluronic acid, such as a combination of collagenase,dispase, and hyaluronidase or a combination of LIBERASE (BoehringerMannheim Corp., Indianapolis, Ind.) and hyaluronidase. Other enzymesknown in the art for cell isolation include papain, deoxyribonucleases,serine proteases, such as trypsin, chymotrypsin, or elastase, that maybe used either on their own or in combination with other enzymes such asmatrix metalloproteases, mucolytic enzymes, and neutral proteases.Serine proteases are preferably used consecutively following use ofother enzymes. The temperature and period of time tissues or cells arein contact with serine proteases is particularly important. Serineproteases may be inhibited by alpha 2 microglobulin in serum andtherefore the medium used for digestion is usually serum-free. EDTA andDNAse are commonly used in enzyme digestion procedures to increase theefficiency of cell recovery. The degree of dilution of the digestion mayalso greatly affect the cell yield as cells may be trapped within theviscous digest.

In some embodiments of the invention, placental tissue is separated intotwo or more sections, each section consisting of either neonatal,neonatal and maternal, or maternal aspect. The separated sections thenare dissociated by mechanical and/or enzymatic dissociation according tothe methods described herein. Cells of neonatal or maternal lineage maybe identified by any means known in the art, for example, by karyotypeanalysis or in situ hybridization for a Y chromosome. Karyotype analysisalso may be used to identify cells of normal karyotype.

Isolated cells or placental tissue from which PDCs grow out may be usedto initiate, or seed, cell cultures. Cells are transferred to steriletissue culture vessels either uncoated or coated with extracellularmatrix or ligands such as laminin, collagen (e.g., native or denatured),gelatin, fibronectin, ornithine, vitronectin, and extracellular membraneprotein (e.g., MATRIGEL (BD Discovery Labware, Bedford, Mass.)). PDCsare cultured in any culture medium capable of sustaining growth of thecells such as, but not limited to, DMEM (high or low glucose), Eagle'sbasal medium, Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove'smodified Dulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM),Liebovitz's L-15 medium, MCDB, DMEM/F12, RPMI 1640, advanced DMEM(Gibco), DMEM/MCDB201 (Sigma), and CELL-GRO FREE. The culture medium maybe supplemented with one or more components including, for example,serum (e.g., fetal bovine serum (FBS), preferably about 2-15% (v/v);equine serum (ES); human serum (HS)); beta-mercaptoethanol (BME),preferably about 0.001% (v/v); one or more growth factors, for example,platelet-derived growth factor (PDGF), epidermal growth factor (EGF),basic fibroblast growth factor (bFGF), insulin-like growth factor-1(IGF-1), leukemia inhibitory factor (LIF), vascular endothelial growthfactor (VEGF), and erythropoietin (EPO); amino acids, includingL-valine; and one or more antibiotic and/or antimycotic agents tocontrol microbial contamination, such as, for example, penicillin G,streptomycin sulfate, amphotericin B, gentamicin, and nystatin, eitheralone or in combination. The culture medium preferably comprises Growthmedium (DMEM-low glucose), serum, BME, an antimycotic agent, and anantibiotic agent).

The cells are seeded in culture vessels at a density to allow cellgrowth. For example, the cells may be seeded at low density (forexample, about 1,000 to about 5,000 cells/cm²) to high density (forexample, about 50,000 or more cells/cm²). In a preferred embodiment, thecells are cultured at about 0 to about 5 percent by volume CO₂ in air.In some preferred embodiments, the cells are cultured at about 2 toabout 25 percent O₂ in air, preferably about 5 to about 20 percent O₂ inair. The cells preferably are cultured at about 25 to about 40° C., morepreferably about 35° C. to about 39° C., and more preferably arecultured at 37° C. The cells are preferably cultured in an incubator.The medium in the culture vessel can be static or agitated, for example,using a bioreactor. PDCs preferably are grown under low oxidative stress(e.g., with addition of glutathione, ascorbic acid, catalase,tocopherol, N-acetylcysteine). “Low oxidative stress”, as used herein,refers to conditions of no or minimal free radical damage to thecultured cells.

Methods for the selection of the most appropriate culture medium, mediumpreparation, and cell culture techniques are well known in the art andare described in a variety of sources, including Doyle et al., (eds.),1995, CELL & TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley & Sons,Chichester; and Ho and Wang (eds.), 1991, ANIMAL CELL BIOREACTORS,Butterworth-Heinemann, Boston, which are incorporated herein byreference.

The culture medium is changed as necessary, for example, by carefullyaspirating the medium from the dish, for example, with a pipette, andreplenishing with fresh medium. Incubation is continued until asufficient number or density of cells accumulate in the dish. Theoriginal explanted tissue sections may be removed and the remainingcells trypsinized using standard techniques or using a cell scraper.After trypsinization, the cells are collected, removed to fresh mediumand incubated as above. In some embodiments, the medium is changed atleast once at approximately 24 hours post-trypsinization to remove anyfloating cells. The cells remaining in culture are considered to bePDCs.

After culturing the cells or tissue fragments for a sufficient period oftime, PDCs will have grown out, either as a result of migration from theplacental tissue or cell division, or both. In some embodiments of theinvention, PDCs are passaged, or removed to a separate culture vesselcontaining fresh medium of the same or a different type as that usedinitially, where the population of cells can be mitotically expanded.PDCs are preferably passaged up to about 100% confluence, morepreferably about 70 to about 85% confluence. The lower limit ofconfluence for passage is understood by one skilled in the art. Thecells of the invention may be used at any point between passage 0 andsenescence. The cells preferably are passaged between about 3 and about25 times, more preferably are passaged about 4 to about 12 times, andpreferably are passaged 10 or 11 times. Cloning and/or subcloning may beperformed to confirm that a clonal population of cells has beenisolated.

Cells of the invention may be cryopreserved. PDCs are preferablycryopreserved in cryopreservation medium, for example, culture mediumincluding but not limited to Growth medium, or cell freezing medium, forexample commercially available cell freezing medium, such as but notlimited to C2695 (Sigma), C2639 (Sigma), or C6039 (Sigma). Thecryopreservation medium preferably comprises dimethylsulfoxide (DMSO),for example about 10% (v/v). The cryopreservation medium may compriseadditional cryopreservation agents including but not limited tomethylcellulose and/or glycerol. The cells are preferably cooled atabout 1° C./min. The preferred cryopreservation temperature is about−80° C. to about −180° C., more preferably is about −90° C. to about−160° C., and most preferably is about −125 to about −140° C.Cryopreserved cells preferably are transferred to liquid nitrogen priorto thawing for use. In some embodiments, for example, once the ampouleshave reached about −90° C., they are transferred to a liquid nitrogenstorage area. Cryopreserved cells preferably are thawed at a temperatureof about 25° C. to about 40° C., more preferably about 35° C. to about39° C., and more preferably about 37° C.

Characterization of PDCs

PDCs may be characterized, for example, by growth characteristics (e.g.,population doubling capability, doubling time, passages to senescence),karyotype analysis (e.g., maternal or neonatal lineage), flow cytometry(e.g., FACS analysis), immunohistochemistry and/or immunocytochemistry(e.g., for detection of epitopes including but not limited to vimentin,desmin, alpha-smooth muscle actin, cytokeratin 18, von Willebrandfactor, CD34, GROalpha, GCP-2, oxidized low density lipoprotein receptor1, and NOGO-A), gene expression profiling (e.g., gene chip arrays;polymerase chain reaction (for example, reverse transcriptase PCR, realtime PCR, and conventional PCR)), protein arrays, protein secretion(e.g., by plasma clotting assay or analysis of PDC-conditioned medium,for example, by Enzyme Linked ImmunoSorbent Assay (ELISA)), antibodyanalysis (e.g., ELISA, antibody staining for cell surface markersincluding but not limited to CD10, CD13, CD31, CD34, CD44, CD45, CD73,CD80, CD86, CD90, CD117, CD141, CD178, platelet-derived growth factorreceptor alpha (PDGFr-alpha), HLA class I antigens (HLA-A, HLA-B,HLA-C), HLA class II antigens (HLA-DP, HLA-DQ, HLA-DR), B7-H2, andPD-L2), mixed lymphocyte reaction (e.g., as measure of stimulation ofallogeneic peripheral blood mononuclear cells (PBMCs), for example,allogeneic lymphocytes, e.g., naïve CD4+ T cells), or other methodsknown in the art.

The placenta-derived cells of the invention preferably are derived fromhuman postpartum placenta tissue substantially free of blood. PDCs arecapable of self-renewal and expansion in culture and have the potentialto differentiate into cells of another phenotype. PDCs require L-valinefor growth. PDCs preferably are capable of growth in about 5% to about20% oxygen. PDCs preferably comprise at least one of the followingcharacteristics:

(a) production of at least one of tissue factor, vimentin, granulocytechemotactic protein-2 (GCP-2), and alpha-smooth muscle actin;

(b) lack of production of at least one of GRO-alpha and oxidized lowdensity lipoprotein receptor, as detected by flow cytometry;

(c) production of at least one of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, PD-L2 and HLA-A,B,C;

(d) lack of production of at least one of CD31, CD34, CD45, CD80, CD86,CD117, CD141, CD178, B7-H2, HLA-G, and HLA-DP, DQ, DR, as detected byflow cytometry;

(e) expression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an iliac crest bone marrow cell, is increasedfor at least one of C-type lectin superfamily member A2, Wilms tumor 1,aldehyde dehydrogenase 1 family member A2, renin, oxidized low densitylipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671,hypothetical protein DKFZp564F013, downregulated in ovarian cancer 1,and clone DKFZp547K1113;

(f) expression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an iliac crest bone marrow cell, is reducedfor at least one of: short stature homeobox 2; heat shock 27 kDa protein2; chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);elastin; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeobox 2; sine oculis homeobox homolog 1; crystallin, alpha B;dishevelled associated activator of morphogenesis 2; DKFZP586B2420protein; similar to neuralin 1; tetranectin; src homology three (SH3)and cysteine rich domain; B-cell translocation gene 1,anti-proliferative; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; hypothetical protein FLJ23191; interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog7; hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C;iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; cDNA FLJ12280 fis, clone MAMMA1001744; cytokinereceptor-like factor 1; potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 4; integrin, alpha 7;DKFZP586L151 protein; transcriptional co-activator with PDZ-bindingmotif (TAZ); sine oculis homeobox homolog 2; KIAA1034 protein; earlygrowth response 3; distal-less homeobox 5; hypothetical proteinFLJ20373; aldo-keto reductase family 1, member C3 (3-alphahydroxysteroid dehydrogenase, type II); biglycan; fibronectin 1;proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains);cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; cDNA DKFZp564B222 (fromclone DKFZp564B222); vesicle-associated membrane protein 5;EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; and insulin-like growthfactor binding protein 2, 36 kDa;

(g) secretion of at least one of monocyte chemotactic protein 1 (MCP-1),interleukin-6 (IL-6), stromal-derived factor 1alpha (SDF-1alpha),interleukin 8 (IL8), granulocyte chemotactic protein-2 (GCP-2),hepatocyte growth factor (HGF), keratinocyte growth factor (KGF),heparin-binding epidermal growth factor (HB-EGF), brain-derivedneurotrophic factor (BDNF), tissue inhibitor of matrix metalloproteinase1 (TIMP1), thrombopoietin (TPO), macrophage inflammatory protein 1alpha(MIP1a), Rantes (regulated on activation, normal T cell expressed andsecreted), thymus and activation-regulated chemokine (TARC), andEotaxin;

(h) lack of secretion of at least one of fibroblast growth factor (FGF),vascular endothelial growth factor (VEGF), angiopoietin 2 (ANG2),platelet derived growth factor (PDGF-bb), transforming growth factorbeta2 (TGFbeta2), macrophage inflammatory protein 1beta (MIP1b), 1309,and macrophage-derived chemokine (MDC), as detected by ELISA; and

(i) the ability to undergo at least 40 population doublings in culture.

Population doubling may be calculated as [ln(cell final/cell initial)/ln2]. Doubling time may be calculated as (time in culture (h)/populationdoubling).

In preferred embodiments, the cell comprises two or more of theforegoing characteristics. More preferred are those cells comprisingthree, four, or five or more of the characteristics. Still morepreferred are those postpartum-derived cells comprising six, seven, oreight or more of the characteristics. Still more preferred are thosecells comprising all nine of the claimed characteristics.

Also presently preferred are cells that produce at least two of GCP-2,tissue factor, vimentin, and alpha-smooth muscle actin. More preferredare those cells producing three or four of the proteins GCP-2, tissuefactor, vimentin, and alpha-smooth muscle actin.

In some embodiments, the cells of the invention do not produce at leastone of oxidized low density lipoprotein receptor or GRO-alpha, asdetected by FACS analysis. In some embodiments, the cells produceneither protein as detected by FACS analysis.

The skilled artisan will appreciate that cell markers are subject tovary somewhat under vastly different growth conditions, and thatgenerally herein described are characterizations in Growth Medium, orvariations thereof. Postpartum-derived cells that produce of at leastone, two, three, or four of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha,PD-L2 and HLA-A,B,C are preferred. More preferred are those cellsproducing five, six, or seven of these cell surface markers. Still morepreferred are postpartum-derived cells that can produce all eight of theforegoing cell surface marker proteins.

PPDCs that lack of production of at least one, two, three, four of theproteins CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2,HLA-G, and HLA-DR,DP,DQ, as detected by flow cytometry are preferred.PPDCs lacking production of at least five, six, seven or eight or moreof these markers are preferred. More preferred are cells which lackproduction of at least nine or ten of the cell surface markers. Mosthighly preferred are those cells lacking production of eleven, twelve,or thirteen of the foregoing identifying proteins.

Presently preferred cells produce each of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, and HLA-A,B,C, and do not produce any of CD31, CD34, CD45,CD117, CD141, or HLA-DR,DP,DQ, as detected by flow cytometry.

It is preferred that postpartum-derived cells exhibit increasedexpression, relative to a human cell that is a fibroblast, a mesenchymalstem cell, or an iliac crest bone marrow cell, for at least one, two, orthree of C-type lectin superfamily member A2, Wilms tumor 1, aldehydedehydrogenase 1 family member A2, renin, oxidized low densitylipoprotein receptor 1, protein kinase C zeta, clone IMAGE:4179671,hypothetical protein DKFZp564F013, downregulated in ovarian cancer 1,and clone DKFZp547K1113. More preferred are those cells which exhibitincreased expression for four, five, six, or seven, and still morepreferred are cells capable of increased expression of eight, nine, orten of the foregoing genes.

For some embodiments, preferred are cells, which relative to a humancell that is a fibroblast, a mesenchymal stem cell, or an iliac crestbone marrow cell, have reduced expression for at least one of the genescorresponding to: short stature homeobox 2; heat shock 27 kDa protein 2;chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);elastin; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeobox 2; sine oculis homeobox homolog 1; crystallin, alpha B;dishevelled associated activator of morphogenesis 2; DKFZP586B2420protein; similar to neuralin 1; tetranectin; src homology three (SH3)and cysteine rich domain; B-cell translocation gene 1,anti-proliferative; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; hypothetical protein FLJ23191; interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer; frizzled homolog7; hypothetical gene BC008967; collagen, type VIII, alpha 1; tenascin C;iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; cDNA FLJ12280 fis, clone MAMMA1001744; cytokinereceptor-like factor 1; potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 4; integrin, alpha 7;DKFZP586L151 protein; transcriptional co-activator with PDZ-bindingmotif (TAZ); sine oculis homeobox homolog 2; KIAA1034 protein; earlygrowth response 3; distal-less homeobox 5; hypothetical proteinFLJ20373; aldo-keto reductase family 1, member C3 (3-alphahydroxysteroid dehydrogenase, type II); biglycan; fibronectin 1;proenkephalin; integrin, beta-like 1 (with EGF-like repeat domains);cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein; natriureticpeptide receptor C/guanylate cyclase C (atrionatriuretic peptidereceptor C); hypothetical protein FLJ14054; cDNA DKFZp564B222 (fromclone DKFZp564B222); vesicle-associated membrane protein 5;EGF-containing fibulin-like extracellular matrix protein 1;BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);neuroblastoma, suppression of tumorigenicity 1; and insulin-like growthfactor binding protein 2, 36 kDa. More preferred are cells that have,relative to human fibroblasts, mesenchymal stem cells, or iliac crestbone marrow cells, reduced expression of at least 5, 10, 15 or 20 genescorresponding to those listed above. Presently more preferred are cellswith reduced relative expression of at least 25, 30, or 35 of the genescorresponding to the listed sequences. Also more preferred are thosepostpartum-derived cells having expression that is reduced, relative tothat of a human fibroblast, a mesenchymal stem cell, or an iliac crestbone marrow cell, of genes corresponding to 35 or more, 40 or more, oreven all of the sequences listed.

Secretion of certain growth factors and other cellular proteins can makecells of the invention particularly useful. Preferred placenta-derivedcells secrete at least one, two, three, or four of monocyte chemotacticprotein 1 (MCP-1), interleukin-6 (IL-6), stromal-derived factor 1alpha(SDF-1alpha), interleukin 8 (IL8), granulocyte chemotactic protein-2(GCP-2), hepatocyte growth factor (HGF), keratinocyte growth factor(KGF), heparin-binding epidermal growth factor (HB-EGF), brain-derivedneurotrophic factor (BDNF), tissue inhibitor of matrix metalloproteinase1 (TIMP1), thrombopoietin (TPO), macrophage inflammatory protein 1alpha(MIP1a), Rantes (regulated on activation, normal T cell expressed andsecreted), thymus and activation-regulated chemokine (TARC), andEotaxin. Cells which secrete more than five, six, seven or eight of thelisted proteins are also useful and preferred. Cells which can secreteat least nine, ten, eleven or more of the factors are more preferred, asare cells which can secrete twelve thirteen, or fourteen, or even all ofthe proteins in the foregoing list.

While secretion of such factors is useful, PDCs can also becharacterized by their lack of secretion of factors into the medium.Postpartum-derived cells that lack secretion of at least one, two,three, or four of fibroblast growth factor (FGF), vascular endothelialgrowth factor (VEGF), angiopoietin 2 (ANG2), platelet derived growthfactor (PDGF-bb), transforming growth factor beta2 (TGFbeta2),macrophage inflammatory protein 1beta (MIP1b), 1309, andmacrophage-derived chemokine (MDC), as detected by ELISA, are preferredfor use. Cells that are characterized in their lack secretion of five,six, or seven of the foregoing proteins are more preferred. Cells whichlack secretion of all of the factors listed above are also preferred.

Examples of placenta-derived cells of the invention were deposited withthe American Type Culture Collection (ATCC, Manassas, Va.) and assignedATCC Accession Numbers as follows: (1) strain designation PLA 071003(P8) was deposited Jun. 15, 2004 and assigned Accession No. PTA-6074;(2) strain designation PLA 071003 (P11) was deposited Jun. 15, 2004 andassigned Accession No. PTA-6075; and (3) strain designation PLA 071003(P16) was deposited Jun. 16, 2004 and assigned Accession No. PTA-6079.

Examples of umbilical cord-derived cells of the invention were depositedwith the American Type Culture Collection (ATCC, Manassas, Va.) on Jun.10, 2004, and assigned ATCC Accession Numbers as follows: (1) straindesignation UMB 022803 (P7) was assigned Accession No. PTA-6067; and (2)strain designation UMB 022803 (P17) was assigned Accession No. PTA-6068.

PDCs of the invention may be isolated. The invention providescompositions of PDCs, including populations of PDCs. In someembodiments, the population is heterogeneous. A heterogeneous cellpopulation of the invention may comprise at least about 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% PDCs of the invention. Theheterogeneous cell populations of the invention may further comprisestem cells or progenitor cells. In some embodiments, the population issubstantially homogeneous, i.e., comprises substantially only PPDCs(preferably at least about 96%, 97%, 98%, 99% or more PPDCs). Thehomogeneous cell population of the invention may comprise neonatalplacenta-derived cells or maternal placenta-derived cells. Homogeneityof a cell population may be achieved by any method known in the art, forexample, by cell sorting (e.g., flow cytometry), bead separation, or byclonal expansion.

The cells of the invention can be induced to differentiate to cells ofmesodermal, ectodermal, or endodermal phenotype or lineage.

Culture of PDCs in a Chondrogenic Medium

PDCs may be induced to differentiate into a chondrogenic lineage bysubjecting them to differentiation-inducing cell culture conditions. Insome embodiments, PDCs may be induced to differentiate to a chondrogeniclineage by, for example, contacting PDCs with specific exogenous growthfactors (e.g., in culture), such as, for example, one or more of GDF-5or transforming growth factor beta3 (TGF-beta3), with or withoutascorbate.

Preferred chondrogenic medium is supplemented with an antibiotic agent,amino acids including proline and glutamine, sodium pyruvate,dexamethasone, ascorbic acid, and insulin/tranferrin/selenium.Chondrogenic medium is preferably supplemented with sodium hydroxideand/or collagen. Most preferably, chondrogenic culture medium issupplemented with collagen. The cells may be cultured at high or lowdensity. Cells are preferably cultured in the absence of serum.

Chondrogenic differentiation may be assessed, for example, by Safranin-Ostaining for glycosaminoglycan expression or hematoxylin/eosin staining.

Culture of PDCs in an Adipogenic Medium

PDCs may be induced to differentiate into an adipogenic lineagephenotype by subjecting them to differentiation-inducing cell cultureconditions. In some embodiments, PDCs are cultured in a defined mediumfor inducing differentiation to an adipogenic lineage. Examples ofadipogenic media include, but are not limited to, media containing oneor more glucocorticoids (e.g., dexamethasone, indomethasone,hydrocortisone, cortisone), insulin, a compound which elevatesintracellular levels of cAMP (e.g., dibutyryl-cAMP; 8-CPT-cAMP(8-(4)chlorophenylthio)-adenosine, 3′,5′ cyclic monophosphate);8-bromo-cAMP; dioctanoyl-cAMP; forskolin) and/or a compound whichinhibits degradation of cAMP (e.g., a phosphodiesterase inhibitor suchas isobutylmethylxanthine (IBMX), methyl isobutylxanthine, theophylline,caffeine, indomethacin), and serum.

Adipogenesis may be assessed by Oil-Red-O staining to determine thepresence of lipid droplet formation or by detecting the expression ofPPAR gamma or leptin.

Culture of PDCs in an Osteogenic Medium

PDCs may be induced to differentiate into an osteogenic lineagephenotype by subjecting them to differentiation-inducing cell cultureconditions. In some embodiments, PDCs are cultured in osteogenic mediumsuch as, but not limited to, media (e.g., DMEM-low glucose) containingabout 10⁻⁷ molar and about 10⁻⁹ molar dexamethasone in combination withabout 10 micromolar to about 50 micromolar ascorbate phosphate salt(e.g., ascorbate-2-phosphate) and between about 10 nanomolar and about10 millimolar beta-glycerophosphate. The medium preferably includesserum (e.g., bovine serum, horse serum). Osteogenic medium also maycomprise one or more antibiotic/antimycotic agents. The osteogenicmedium is preferably supplemented with transforming growth factor-beta(e.g., TGF-beta1) and/or bone morphogenic protein (e.g., BMP-2, BMP-4,or a combination thereof; most preferably BMP-4)

Cells may be analyzed for an osteogenic phenotype by any method known inthe art, e.g., von Kossa staining or by detection of osteogenic markerssuch as osteocalcin, bone sialoprotein, or alkaline phosphatase.

Culture of PDCs in Neurogenic Medium

PDCs may be induced to differentiate into a neural lineage phenotype bysubjecting them to differentiation-inducing cell culture conditions.This may be accomplished by one or more methods known in the art. Forinstance, as exemplified herein, PDCs may be cultured in a neurogenicmedium such as a serum-free DMEM/F12 composition containing butylatedhydroxanisole, potassium chloride, insulin, forskolin, valproic acid,and hydrocortisone.

Alternatively, PDCs may be plated on flasks coated with laminin inNeurobasal-A medium (Invitrogen, Carlsbad, Calif.) containing B27 (B27supplement, Invitrogen), L-glutamine and Penicillin/Streptomycin, thecombination of which is referred to herein as Neural ProgenitorExpansion (NPE) media. NPE media may be further supplemented with bFGFand/or EGF.

Alternatively, PDCs may be induced to differentiate in vitro by (1)co-culturing the PDCs with neural progenitor cells, or (2) growing thePDCs in neural progenitor cell-conditioned medium.

Differentiation of the PDCs to a neurogenic lineage may be demonstratedby a bipolar cell morphology with extended processes. The induced cellpopulations may stain positive for the presence of nestin.Differentiated PDCs may be assessed by detection of nestin, TuJ1 (BIIItubulin), GFAP, tyrosine hydroxylase, O4, GABA, and myelin basic protein(MBP). In some embodiments, PDCs have the ability to formthree-dimensional bodies characteristic of neural stem cell formation ofneurospheres.

Assessment of Differentiation

PDCs may be induced to differentiate to an ectodermal, endodermal, ormesodermal lineage. Methods to characterize differentiated cells thatdevelop from the PDCs of the invention, include, but are not limited to,histological, morphological, biochemical and immunohistochemicalmethods, or using cell surface markers, or genetically or molecularly,or by identifying factors secreted by the differentiated cell, and bythe inductive qualities of the differentiated PDCs.

Methods of Using PDCs or Components or Products Thereof

Genetic Engineering of PDCs

The cells of the invention can be engineered to express a therapeuticprotein using any of a variety of vectors including, but not limited to,integrating viral vectors, e.g., retrovirus vector or adeno-associatedviral vectors; non-integrating replicating vectors, e.g., papillomavirus vectors, SV40 vectors, adenoviral vectors; orreplication-defective viral vectors. Other methods of introducing DNAinto cells include the use of liposomes, electroporation, a particlegun, or by direct DNA injection.

Hosts cells are preferably transformed or transfected with DNAcontrolled by or in operative association with, one or more appropriateexpression control elements such as promoter or enhancer sequences,transcription terminators, polyadenylation sites, among others, and aselectable marker.

Following the introduction of the foreign DNA, engineered cells may beallowed to grow in enriched media and then switched to selective media.The selectable marker in the foreign DNA confers resistance to theselection and allows cells to stably integrate the foreign DNA as, forexample, on a plasmid, into their chromosomes and grow to form fociwhich, in turn, can be cloned and expanded into cell lines.

This method can be advantageously used to engineer cell lines whichexpress the gene product.

Any promoter may be used to drive the expression of the inserted gene.For example, viral promoters include, but are not limited to, the CMVpromoter/enhancer, SV40, papillomavirus, Epstein-Barr virus or elastingene promoter. Preferably, the control elements used to controlexpression of the gene of interest should allow for the regulatedexpression of the gene so that the product is synthesized only whenneeded in vivo. If transient expression is desired, constitutivepromoters are preferably used in a non-integrating and/orreplication-defective vector. Alternatively, inducible promoters couldbe used to drive the expression of the inserted gene when necessary.

Inducible promoters include, but are not limited to, those associatedwith metallothionein and heat shock proteins.

Examples of transcriptional control regions that exhibit tissuespecificity which have been described and could be used include but arenot limited to: elastase I gene control region, which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38:639; Ornitz et al.,1985, Cold Spring Harbor Symp. Quant. Biol. 50:399; MacDonald, 1987,Hepatology 7:42S-51S); insulin gene control region, which is active inpancreatic beta cells (Hanahan, 1985, Nature 315:115); myelin basicprotein gene control region, which is active in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703); myosin light chain-2gene control region, which is active in skeletal muscle (Shani, 1985,Nature 314:283); and gonadotropic releasing hormone gene control region,which is active in the hypothalamus (Mason et al., 1986, Science234:1372).

The cells of the invention may be genetically engineered to “knock out”or “knock down” expression of factors that promote inflammation orrejection at the implant site. Negative modulatory techniques for thereduction of target gene expression levels or target gene productactivity levels are discussed below. “Negative modulation,” as usedherein, refers to a reduction in the level and/or activity of targetgene product relative to the level and/or activity of the target geneproduct in the absence of the modulatory treatment. The expression of agene native to a cell can be reduced or knocked out using a number oftechniques including, for example, inhibition of expression byinactivating the gene completely (commonly termed “knockout”) using thehomologous recombination technique. Usually, an exon encoding animportant region of the protein (or an exon 5′ to that region) isinterrupted by a positive selectable marker, e.g., neo, preventing theproduction of normal mRNA from the target gene and resulting ininactivation of the gene. A gene may also be inactivated by creating adeletion in part of a gene or by deleting the entire gene. By using aconstruct with two regions of homology to the target gene that are farapart in the genome, the sequences intervening the two regions can bedeleted (Mombaerts et al., 1991, Proc. Nat. Acad. Sci. U.S.A. 88:3084).

Antisense, DNAzymes, small interfering RNA, and ribozyme molecules whichinhibit expression of the target gene can also be used in accordancewith the invention to reduce the level of target gene activity. Forexample, antisense RNA molecules which inhibit the expression of majorhistocompatibility gene complexes (HLA) have been shown to be mostversatile with respect to immune responses. Still further, triple helixmolecules can be utilized in reducing the level of target gene activity.

These techniques are described in detail by L. G. Davis et al. (eds),1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2nd ed., Appleton & Lange,Norwalk, Conn., which is incorporated herein by reference.

Using any of the foregoing techniques, for example, the expression ofIL-1 can be knocked out or knocked down in the cells of the invention toreduce the production of inflammatory mediators by the cells of theinvention. Likewise, the expression of MEW class II molecules can beknocked out or knocked down in order to reduce the risk of rejection ofthe implanted tissue.

Once the cells of the invention have been genetically engineered, theymay be directly implanted into the patient.

Alternatively, the genetically engineered cells may be used to producenew tissue in vitro, which is then implanted in the subject.

Secretion of Trophic Factors by PDCs

The secretion of growth factors by PDCs may provide trophic support fora second cell type in vitro or in vivo. PDCs may secrete, for example,interleukin 8 (IL8), tissue factor, hepatocyte growth factor (HGF),monocyte chemotactic protein 1 (MCP-1), keratinocyte growth factor(KGF), tissue inhibitor of matrix metalloproteinase 1 (TIMP1),thrombopoietin (TPO), heparin-binding epidermal growth factor (HB-EGF),stromal-derived factor 1alpha (SDF-1alpha), brain-derived neurotrophicfactor (BDNF), interleukin-6 (IL-6), granulocyte chemotactic protein-2(GCP-2), macrophage inflammatory protein 1alpha (MIP1a), monocytechemoattractant-1 (MCP-1), Rantes (regulated on activation, normal Tcell expressed and secreted), thymus and activation-regulated chemokine(TARC), Eotaxin, NGF, NT-3, IL-7, IL-1, SCF, AMPS, or Cystatin-C insubstantially homogeneous populations of cells, which can be augmentedby a variety of techniques, including ex vivo cultivation of the cellsin chemically defined medium.

In some aspects of the invention, a population of PDCs supports thesurvival, proliferation, growth, maintenance, maturation,differentiation, or increased activity of cells including stem cells,such as neural stem cells (NSC), hematopoietic stem cells (HPC,particularly CD34+ stem cells), embryonic stem cells (ESC), and mixturesthereof. In other embodiments, the population supported by the PDCs issubstantially homogeneous, substantially homogeneous, i.e., comprisessubstantially only PDCs (preferably at least about 96%, 97%, 98%, 99% ormore PDCs).

Conditioned Medium of PDCs

Another embodiment of the invention features use of PDCs for productionof conditioned medium, either from undifferentiated PDCs or from PDCsincubated under conditions that stimulate differentiation into a givenlineage. Such conditioned media are contemplated for use in in vitro orex vivo culture of cells, for example, stem or progenitor cells, or foruse in vivo, for example, to support transplanted cells (e.g.,homogeneous or heterogeneous populations of PDCs).

Co-Cultures of PDCs with Other Cell Types

PDCs have the ability to support survival, growth, and differentiationof other cell types in co-culture. Accordingly, in another embodiment,PDCs are co-cultured in vitro to provide trophic support to other cells.For co-culture, it may be desirable for the PDCs and the desired othercells to be co-cultured under conditions in which the two cell types arein contact. This can be achieved, for example, by seeding the cells as aheterogeneous population of cells in culture medium or onto a suitableculture substrate. Alternatively, the PDCs can first be grown toconfluence and employed as a substrate for the second desired cell typein culture. In this latter embodiment, the cells may further bephysically separated, e.g., by a membrane or similar device, such thatthe other cell type may be removed and used separately following theco-culture period. Use of PDCs in co-culture to promote expansion anddifferentiation of other cell types may find applicability in researchand in clinical/therapeutic areas. For instance, PDC co-culture may beutilized to facilitate growth and differentiation of cells of a givenphenotype in culture, for basic research purposes or for use in drugscreening assays, for example. PDC co-culture may also be utilized forex vivo expansion of cells of a given phenotype for later administrationfor therapeutic purposes. For example, cells may be harvested from anindividual, expanded ex vivo in co-culture with PDCs, then returned tothat individual (autologous transfer) or another individual (syngeneicor allogeneic transfer). In these embodiments, it will be appreciatedthat, following ex vivo expansion, the mixed population of cellscomprising the PDCs could be administered to a patient in need oftreatment. Alternatively, in situations where autologous transfer isappropriate or desirable, the co-cultured cell populations may bephysically separated in culture, enabling removal of the autologouscells for administration to the patient.

Cell Therapy

As demonstrated herein, PDCs have been shown to be effectivelytransplanted into the body and to supply lost function in animal modelsaccepted for predictability of efficacy in humans. These results supporta preferred embodiment of the invention, wherein PDCs are used in celltherapy for treating a condition, injury, or disease. For example, PDCsof the invention may be used to treat patients requiring the repair orreplacement of a tissue or organ resulting from disease or trauma orfailure of the tissue to develop normally, or to provide a cosmeticfunction, such as to augment features of the body. Once transplantedinto a target location in the body, PDCs may themselves differentiateinto one or more phenotypes, or they may provide trophic support forother cell types in vivo, or they may exert a beneficial effect in bothof those fashions, among others.

PDCs may be administered alone (e.g., as substantially homogeneouspopulations) or as admixtures with other cells. PDCs may be administeredas formulated in a pharmaceutical preparation with a matrix, or withconventional pharmaceutically acceptable carriers. Where PDCs areadministered with other cells, they may be administered simultaneouslyor sequentially with the other cells (either before or after the othercells). Cells that may be administered in conjunction with PDCs include,but are not limited to, other multipotent or pluripotent cells. Thecells of different types may be admixed with the PDCs immediately orshortly prior to administration, or they may be co-cultured together fora period of time prior to administration.

PDCs may be administered with other beneficial drugs or biologicalmolecules (growth factors, trophic factors). When PDCs are administeredwith other agents, they may be administered together in a singlepharmaceutical composition, or in separate pharmaceutical compositions,simultaneously or sequentially with the other bioactive factors (eitherbefore or after administration of the other agents). Examples ofbioactive factors include anti-apoptotic agents (e.g., EPO, EPOmimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors);anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-betainhibitors, statins, IL-6 and IL-1 inhibitors, PEMIROLAST, TRANILAST,REMICADE, SIROLIMUS, and NSAIDs (non-steroidal anti-inflammatory drugs;e.g., TEPDXALIN, TOLMETIN, SUPROFEN); immunosupressive/immunomodulatoryagents (e.g., calcineurin inhibitors, such as cyclosporine, tacrolimus;mTOR inhibitors (e.g., SIROLIMUS, EVEROLIMUS); anti-proliferatives(e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g.,prednisolone, hydrocortisone); antibodies such as monoclonalanti-IL-2Ralpha receptor antibodies (e.g., basiliximab, daclizumab),polyclonal anti-T-cell antibodies (e.g., anti-thymocyte globulin (ATG);anti-lymphocyte globulin (ALG); monoclonal anti-T cell antibody OKT3));anti-thrombogenic agents (e.g., heparin, heparin derivatives, urokinase,PPack (dextrophenylalanine proline arginine chloromethylketone),antithrombin compounds, platelet receptor antagonists, anti-thrombinantibodies, anti-platelet receptor antibodies, aspirin, dipyridamole,protamine, hirudin, prostaglandin inhibitors, and platelet inhibitors);and anti-oxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol,coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine). Drugs whichmay be co-administered include local anesthetics. As another example,the cells may be co-administered with scar inhibitory factor asdescribed in U.S. Pat. No. 5,827,735, incorporated herein by reference.

In one embodiment, PDCs are administered as undifferentiated cells,i.e., as cultured in Growth Medium. Alternatively, PDCs may beadministered following exposure in culture to conditions that stimulatedifferentiation toward a desired phenotype.

The cells of the invention may be surgically implanted, injected,delivered (e.g., by way of a catheter or syringe), or otherwiseadministered directly or indirectly to the site in need of repair oraugmentation. Routes of administration of the cells of the invention orcompositions thereof include, but are not limited to, oral, nasal,intraarterial, parenteral, intravenous, ophthalmic, intramuscular,subcutaneous, intraperitoneal, intracerebral, intraventricular,intracerebroventricular, intrathecal, intracisternal, intraspinal and/orperi-spinal routes of administration by delivery via intracranial orintravertebral needles and/or catheters with or without pump devices.

When cells are administered in semi-solid or solid devices, surgicalimplantation into a precise location in the body is typically a suitablemeans of administration. Liquid or fluid pharmaceutical compositions,however, may be administered to a more general location (e.g.,throughout a diffusely affected area, for example), from which theymigrate to a particular location, e.g., by responding to chemicalsignals.

Other embodiments encompass methods of treatment by administeringpharmaceutical compositions comprising PDC cellular components (e.g.,cell lysates or components thereof) or products (e.g., extracellularmatrix, trophic and other biological factors produced naturally by PDCsor through genetic modification, conditioned medium from PDC culture).Again, these methods may further comprise administering bioactivefactors, such as anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO,IGF-I and IGF-II, HGF, caspase inhibitors); anti-inflammatory agents(e.g., p38 MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and NSAIDs(non-steroidal anti-inflammatory drugs; e.g., TEPDXALIN, TOLMETIN,SUPROFEN); immunosupressive/immunomodulatory agents (e.g., calcineurininhibitors, such as cyclosporine, tacrolimus; mTOR inhibitors (e.g.,SIROLIMUS, EVEROLIMUS); anti-proliferatives (e.g., azathioprine,mycophenolate mofetil); corticosteroids (e.g., prednisolone,hydrocortisone); antibodies such as monoclonal anti-IL-2Ralpha receptorantibodies (e.g., basiliximab, daclizumab), polyclonal anti-T-cellantibodies (e.g., anti-thymocyte globulin (ATG); anti-lymphocyteglobulin (ALG); monoclonal anti-T cell antibody OKT3));anti-thrombogenic agents (e.g., heparin, heparin derivatives, urokinase,PPack (dextrophenylalanine proline arginine chloromethylketone),antithrombin compounds, platelet receptor antagonists, anti-thrombinantibodies, anti-platelet receptor antibodies, aspirin, dipyridamole,protamine, hirudin, prostaglandin inhibitors, and platelet inhibitors);and anti-oxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol,coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine), localanesthetics, and scar inhibitory factor as described in U.S. Pat. No.5,827,735, incorporated herein by reference.

Dosage forms and regimes for administering PDCs or any of the otherpharmaceutical compositions described herein are developed in accordancewith good medical practice, taking into account the condition of theindividual patient, e.g., nature and extent of the condition beingtreated, age, sex, body weight and general medical condition, and otherfactors known to medical practitioners. Thus, the effective amount of apharmaceutical composition to be administered to a patient is determinedby these considerations as known in the art.

In some embodiments of the invention, it may not be necessary ordesirable to immunosuppress a patient prior to initiation of celltherapy with PDCs. In addition, PDCs have been shown not to stimulateallogeneic PBMCs (for example, allogeneic lymphocytes, e.g., naïve CD4+T cells) in a mixed lymphocyte reaction. Accordingly, transplantationwith allogeneic, or even xenogeneic, PDCs may be tolerated in someinstances.

However, in other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy. This may be accomplished through the use of systemic or localimmunosuppressive agents, or it may be accomplished by delivering thecells in an encapsulated device. PDCs may be encapsulated in a capsulethat is permeable to nutrients and oxygen required by the cell andtherapeutic factors the cell is yet impermeable to immune humoralfactors and cells. Preferably the encapsulant is hypoallergenic, iseasily and stably situated in a target tissue, and provides addedprotection to the implanted structure. These and other means forreducing or eliminating an immune response to the transplanted cells areknown in the art. As an alternative, PDCs may be genetically modified toreduce their immunogenicity.

Survival of transplanted PDCs in a living patient can be determinedthrough the use of a variety of scanning techniques, e.g., computerizedaxial tomography (CAT or CT) scan, magnetic resonance imaging (MRI) orpositron emission tomography (PET) scans. Determination of transplantsurvival can also be done post mortem by removing the target tissue, andexamining it visually or through a microscope. Alternatively, cells canbe treated with stains that are specific for cells of a specificlineage. Transplanted cells can also be identified by priorincorporation of tracer dyes such as rhodamine- or fluorescein-labeledmicrospheres, fast blue, bisbenzamide, ferric microparticles, orgenetically introduced reporter gene products, such asbeta-galactosidase or beta-glucuronidase.

Functional integration of transplanted PDCs into a subject can beassessed by examining restoration of the function that was damaged ordiseased or augmentation of function.

Compositions and Pharmaceutical Compositions

Compositions of PDCs and related products (e.g., extracellular matrix,lysate, cell lysate, conditioned medium), including for examplepharmaceutical compositions, are included within the scope of theinvention. Compositions of the invention may include one or morebioactive factors, for example but not limited to a growth factor, adifferentiation-inducing factor, a cell survival factor such as caspaseinhibitor, an anti-inflammatory agent such as p38 kinase inhibitor, oran angiogenic factor such as VEGF or bFGF. Some examples of bioactivefactors include PDGF-bb, EGF, FGF, IGF, and LIF. In some embodiments,undifferentiated or differentiation-induced PDCs are cultured in contactwith the bioactive factor. In some embodiments, undifferentiated PDCsremain undifferentiated upon contact with the bioactive factor. In otherembodiments, the bioactive factor induces differentiation of the PDCs.

Pharmaceutical compositions of the invention may comprise homogeneous orhetereogeneous populations of differentiated and/or undifferentiatedPDCs, cultures thereof, cell lysates thereof, extracellular matrixproduced thereby, or conditioned medium derived therefrom in apharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers for the cells of the inventioninclude organic or inorganic carrier substances which do notdeleteriously react with the cells of the invention or compositions orcomponents thereof. To the extent they are biocompatible, suitablepharmaceutically acceptable carriers include water, salt solution (suchas Ringer's solution), alcohols, oils, gelatins, and carbohydrates, suchas lactose, amylose, or starch, fatty acid esters,hydroxymethylcellulose, and polyvinyl pyrolidine. Such preparations canbe sterilized, and if desired, mixed with auxiliary agents such aslubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, and coloring.Pharmaceutical carriers suitable for use in the present invention areknown in the art and are described, for example, in PharmaceuticalSciences (17^(th) Ed., Mack Pub. Co., Easton, Pa.) and WO 96/05309, eachof which are incorporated by reference herein.

The dosage (e.g., number of cells to be administered) and frequency ofadministration will depend upon a number of factors, including but notlimited to, the nature of the condition to be treated, the extent of thesymptoms of the condition, characteristics of the patient (e.g., age,size, gender, health).

Use of PDCs for Transplantation

The treatment methods of the subject invention involves the implantationof PDCs into individuals in need thereof. The cells of the presentinvention may be delivered to the site of therapeutic need or “home” tothe site.

The cells of the present invention may differentiate in vivo or providetrophic support to endogenous cells. The appropriate cell implantationdosage in humans can be determined from existing information relatingto, e.g., the activity of the cells. From in vitro culture and in vivoanimal experiments, the amount of factors produced can be quantitated.This information is also useful in calculating an appropriate dosage ofimplanted material. Additionally, the patient can be monitored todetermine if additional implantation can be made or implanted materialreduced accordingly.

To enhance vascularization and survival of the transplanted cells,angiogenic factors such as VEGF, PDGF or bFGF can be added either aloneor in combination with endothelial cells or their precursors includingCD34+, CD34+/CD117+ cells.

One or more other components may be added to transplanted cells,including selected extracellular matrix components, such as one or moretypes of collagen known in the art, and/or growth factors, platelet-richplasma, and drugs. Alternatively, the cells of the invention may begenetically engineered to express and produce growth factors. Bioactivefactors which may be usefully incorporated into the cell formulationinclude anti-apoptotic agents (e.g., EPO, EPO mimetibody, TPO, IGF-I andIGF-II, HGF, caspase inhibitors); anti-inflammatory agents (e.g., p38MAPK inhibitors, TGF-beta inhibitors, statins, IL-6 and IL-1 inhibitors,PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, and NSAIDs (non-steroidalanti-inflammatory drugs; e.g., TEPDXALIN, TOLMETIN, SUPROFEN);immunosupressive/immunomodulatory agents (e.g., calcineurin inhibitors,such as cyclosporine, tacrolimus; mTOR inhibitors (e.g., SIROLIMUS,EVEROLIMUS); anti-proliferatives (e.g., azathioprine, mycophenolatemofetil); corticosteroids (e.g., prednisolone, hydrocortisone);antibodies such as monoclonal anti-IL-2Ralpha receptor antibodies (e.g.,basiliximab, daclizumab), polyclonal anti-T-cell antibodies (e.g.,anti-thymocyte globulin (ATG); anti-lymphocyte globulin (ALG);monoclonal anti-T cell antibody OKT3)); anti-thrombogenic agents (e.g.,heparin, heparin derivatives, urokinase, PPack (dextrophenylalanineproline arginine chloromethylketone), antithrombin compounds, plateletreceptor antagonists, anti-thrombin antibodies, anti-platelet receptorantibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandininhibitors, and platelet inhibitors); and anti-oxidants (e.g., probucol,vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione,L-cysteine, N-acetylcysteine) as well as local anesthetics. As anotherexample, the cells may be co-administered with scar inhibitory factor asdescribed in U.S. Pat. No. 5,827,735, incorporated herein by reference.

Formulation of PDCs for Transplantation

In a non-limiting embodiment, a formulation comprising the cells of theinvention is prepared for injection directly to the site where theproduction of new tissue is desired. For example, and not by way oflimitation, the cells of the invention may be suspended in a hydrogelsolution for injection. Examples of suitable hydrogels for use in theinvention include self-assembling peptides, such as RAD16.Alternatively, the hydrogel solution containing the cells may be allowedto harden, for instance in a mold, to form a matrix having cellsdispersed therein prior to implantation. Or, once the matrix hashardened, the cell formations may be cultured so that the cells aremitotically expanded prior to implantation. The hydrogel is an organicpolymer (natural or synthetic) which is cross-linked via covalent,ionic, or hydrogen bonds to create a three-dimensional open-latticestructure which entraps water molecules to form a gel. Examples ofmaterials which can be used to form a hydrogel include polysaccharidessuch as alginate and salts thereof, peptides, polyphosphazines, andpolyacrylates, which are crosslinked ionically, or block polymers suchas polyethylene oxide-polypropylene glycol block copolymers which arecrosslinked by temperature or pH, respectively. In some embodiments, thesupport for the PDCs of the invention is biodegradable.

In some embodiments of the invention, the formulation comprises an insitu polymerizable gel, as described, for example, in U.S. PatentApplication Publication 2002/0022676; Anseth et al., J. Control Release,78(1-3):199-209 (2002); Wang et al., Biomaterials, 24(22):3969-80(2003).

In some embodiments, the polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

Examples of polymers with basic side groups that can be reacted withanions are poly(vinyl amines), poly(vinyl pyridine), poly(vinylimidazole), and some imino substituted polyphosphazenes. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imino groups.

Alginate can be ionically cross-linked with divalent cations, in water,at room temperature, to form a hydrogel matrix. Due to these mildconditions, alginate has been the most commonly used polymer forhybridoma cell encapsulation, as described, for example, in U.S. Pat.No. 4,352,883 to Lim. In the Lim process, an aqueous solution containingthe biological materials to be encapsulated is suspended in a solutionof a water soluble polymer, the suspension is formed into droplets whichare configured into discrete microcapsules by contact with multivalentcations, then the surface of the microcapsules is crosslinked withpolyamino acids to form a semipermeable membrane around the encapsulatedmaterials.

Polyphosphazenes are polymers with backbones consisting of nitrogen andphosphorous separated by alternating single and double bonds. Eachphosphorous atom is covalently bonded to two side chains.

The polyphosphazenes suitable for cross-linking have a majority of sidechain groups which are acidic and capable of forming salt bridges withdi- or trivalent cations. Examples of preferred acidic side groups arecarboxylic acid groups and sulfonic acid groups. Hydrolytically stablepolyphosphazenes are formed of monomers having carboxylic acid sidegroups that are crosslinked by divalent or trivalent cations such asCa²⁺ or Al³⁺. Polymers can be synthesized that degrade by hydrolysis byincorporating monomers having imidazole, amino acid ester, or glycerolside groups. For example, a polyanionicpoly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized,which is cross-linked with dissolved multivalent cations in aqueousmedia at room temperature or below to form hydrogel matrices.

Biodegradable polyphosphazenes have at least two differing types of sidechains, acidic side groups capable of forming salt bridges withmultivalent cations, and side groups that hydrolyze under in vivoconditions, e.g., imidazole groups, amino acid esters, glycerol andglucosyl.

Hydrolysis of the side chain results in erosion of the polymer. Examplesof hydrolyzing side chains are unsubstituted and substituted imidizolesand amino acid esters in which the group is bonded to the phosphorousatom through an amino linkage (polyphosphazene polymers in which both Rgroups are attached in this manner are known as polyaminophosphazenes).For polyimidazolephosphazenes, some of the “R” groups on thepolyphosphazene backbone are imidazole rings, attached to phosphorous inthe backbone through a ring nitrogen atom. Other “R” groups can beorganic residues that do not participate in hydrolysis, such as methylphenoxy groups or other groups shown in the scientific paper of Allcock,et al., Macromolecule 10:824 (1977). Methods of synthesis of thehydrogel materials, as well as methods for preparing such hydrogels, areknown in the art.

Other components may also be included in the formulation, including butnot limited to any of the following: (1) buffers to provide appropriatepH and isotonicity; (2) lubricants; (3) viscous materials to retain thecells at or near the site of administration, including, for example,alginates, agars and plant gums; and (4) other cell types that mayproduce a desired effect at the site of administration, such as, forexample, enhancement or modification of the formation of tissue or itsphysicochemical characteristics, or is support for the viability of thecells, or inhibition of inflammation or rejection. The cells may becovered by an appropriate wound covering to prevent cells from leavingthe site. Such wound coverings are known as those of skill in the art.

Transplantation of PDCs Using Scaffolds

The cells of the invention or co-cultures thereof may be seeded onto athree-dimensional framework or scaffold and implanted in vivo, where theseeded cells will proliferate on the surface of the framework and form areplacement tissue in vivo in cooperation with the cells of the subject.Such a framework can be implanted in combination with any one or moregrowth factors, cells, drugs or other components described above thatstimulate tissue formation or otherwise enhance or improve the practiceof the invention.

The cells of the invention can be used to produce new tissue in vitro,which can then be implanted, transplanted or otherwise inserted into asite requiring tissue repair, replacement or augmentation in a subject.

In a non-limiting embodiment, the cells of the invention are used toproduce a three-dimensional tissue construct in vitro, which is thenimplanted in vivo. As an example of the production of three-dimensionaltissue constructs, see U.S. Pat. No. 4,963,489, which is incorporatedherein by reference. For example, the cells of the invention may beinoculated or “seeded” onto a three-dimensional framework or scaffold,and proliferated or grown in vitro to form a living tissue that can beimplanted in vivo.

The cells of the invention can be grown freely in a culture vessel tosub-confluency or confluency, lifted from the culture and inoculatedonto a three-dimensional framework. Inoculation of the three-dimensionalframework with a high concentration of cells, e.g., approximately 10⁵ to10⁸ cells per milliliter, will result in the establishment of thethree-dimensional support in relatively shorter periods of time.

Examples of scaffolds which may be used in the present invention includenonwoven mats, porous foams, or self assembling peptides. Nonwoven matsmay, for example, be formed using fibers comprised of a syntheticabsorbable copolymer of glycolic and lactic acids (PGA/PLA), sold underthe tradename VICRYL (Ethicon, Inc., Somerville, N.J.), Foams, composedof, for example, poly(epsilon-caprolactone)/poly(glycolic acid)(PCL/PGA) copolymer, formed by processes such as freeze-drying, orlyophilized, as discussed in U.S. Pat. No. 6,355,699, are also possiblescaffolds. Hydrogels such as self-assembling peptides (e.g., RAD16) mayalso be used. These materials are frequently used as supports for growthof tissue.

The three-dimensional framework also may be made of ceramic materialsincluding, but not limited to: mono-, di-, tri-, alpha-tri-, beta-tri-,and tetra-calcium phosphate, hydroxyapatite, fluoroapatites, calciumsulfates, calcium fluorides, calcium oxides, calcium carbonates,magnesium calcium phosphates, biologically active glasses such asBIOGLASS (University of Florida, Gainesville, Fla.), and mixturesthereof. There are a number of suitable porous biocompatible ceramicmaterials currently available on the commercial market such as SURGIBON(Unilab Surgibone, Inc., Canada), ENDOBON (Merck Biomaterial France,France), CEROS (Mathys, A. G., Bettlach, Switzerland), and INTERPORE(Interpore, Irvine, Calif., United States), and mineralized collagenbone grafting products such as HEALOS (Orquest, Inc., Mountain View,Calif.) and VITOSS, RHAKOSS, and CORTOSS (Orthovita, Malvern, Pa.). Theframework may be a mixture, blend or composite of natural and/orsynthetic materials.

According to a preferred embodiment, the framework is a felt, which canbe composed of a multifilament yarn made from a bioabsorbable material,e.g., PGA, PLA, PCL copolymers or blends, or hyaluronic acid. The yarnis made into a felt using standard textile processing techniquesconsisting of crimping, cutting, carding and needling.

In another preferred embodiment the cells of the invention are seededonto foam scaffolds that may be composite structures. In addition, thethree-dimensional framework may be molded into a useful shape, such asthat of the external portion of the ear, or other specific structure inthe body to be repaired, replaced or augmented.

In some embodiments, the framework is treated prior to inoculation ofthe cells of the invention in order to enhance cell attachment. Forexample, prior to inoculation with the cells of the invention, nylonmatrices could be treated with 0.1 molar acetic acid and incubated inpolylysine, PBS, and/or collagen to coat the nylon. Polystyrene could besimilarly treated using sulfuric acid.

The external surfaces of the three-dimensional framework may be modifiedto improve the attachment or growth of cells and differentiation oftissue, such as by plasma coating the framework or addition of one ormore proteins (e.g., collagens, elastic fibers, reticular fibers),glycoproteins, glycosaminoglycans (e.g., heparin sulfate,chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratinsulfate), a cellular matrix, and/or other materials such as, but notlimited to, gelatin, alginates, agar, agarose, and plant gums, amongothers.

In some embodiments, the scaffold is comprised of or is treated withmaterials that render it non-thrombogenic. These treatments andmaterials may also promote and sustain endothelial growth, migration,and extracellular matrix deposition. Examples of these materials andtreatments include but are not limited to natural materials such asbasement membrane proteins such as laminin and Type IV collagen,synthetic materials such as ePTFE, and segmented polyurethaneureasilicones, such as PURSPAN (The Polymer Technology Group, Inc.,Berkeley, Calif.). These materials can be further treated to render thescaffold non-thrombogenic. Such treatments include anti-thromboticagents such as heparin, and treatments which alter the surface charge ofthe material such as plasma coating.

In some aspects of the invention, it is important to re-create inculture the cellular microenvironment found in vivo, such that theextent to which the cells of the invention are grown prior toimplantation in vivo or use in vitro may vary. In addition, growthfactors may be added to the culture medium prior to, during, orsubsequent to inoculation of the cells to trigger differentiation andtissue formation by the PDCs.

The three-dimensional framework may be modified so that the growth ofcells and the production of tissue thereon is enhanced, or so that therisk of rejection of the implant is reduced. Thus, one or morebiologically active compounds, including, but not limited to,anti-inflammatories, immunosuppressants or growth factors, may be addedto the framework.

Therapeutic Uses for Extracellular Matrix and Cell Lysates Derived fromPDCs

As an alternative to implanting the cells of the invention, or livingtissue produced therefrom, a subject in need of tissue repair,replacement, or augmentation may benefit from the administration of acomponent or product of PDCs, such as the extracellular matrix (ECM) orcell lysate produced by those cells.

In some embodiments, after the cells of the invention have been culturedin vitro, such as, for example, by using a three-dimensional scaffoldsystem described herein, such that a desired amount of ECM has beensecreted onto the framework. Once ECM is secreted onto the framework,the cells may be removed. The ECM may be processed for further use, forexample, as an injectable preparation.

In some embodiments, the cells are killed and cellular debris (e.g.,cellular membranes) is removed from the framework. This process may becarried out in a number of different ways. For example, the livingtissue can be flash-frozen in liquid nitrogen without acryopreservative, or the tissue can be immersed in sterile distilledwater so that the cells burst in response to osmotic pressure. Once thecells have been killed, the cellular membranes may be disrupted andcellular debris removed by treatment with a mild detergent rinse, suchas EDTA, CHAPS or a zwitterionic detergent. An advantage to using a milddetergent rinse is that it solubilizes membrane-bound proteins, whichare often highly antigenic.

Alternatively, the tissue can be enzymatically digested and/or extractedwith reagents that break down cellular membranes. Example of suchenzymes include, but are not limited to, hyaluronidase, dispase,proteases, and nucleases (for example, deoxyribonuclease andribonuclease). Examples of detergents include non-ionic detergents suchas, for example, alkylaryl polyether alcohol (TRITON® X-100),octylphenoxy polyethoxy-ethanol (Rohm and Haas Philadelphia, Pa.),BRIJ-35, a polyethoxyethanol lauryl ether (Atlas Chemical Co., SanDiego, Calif.), polysorbate 20 (TWEEN 20®), a polyethoxyethanol sorbitanmonolaureate (Rohm and Haas), polyethylene lauryl ether (Rohm and Haas);and ionic detergents such as, for example, sodium dodecyl sulphate,sulfated higher aliphatic alcohols, sulfonated alkanes and sulfonatedalkylarenes containing 7 to 22 carbon atoms in a branched or unbranchedchain.

The scaffold comprising the ECM may be used therapeutically as describedabove. Alternatively, ECM may be collected from the scaffold. Collectionof ECM can be accomplished in a variety of ways, depending, for example,on whether the framework is biodegradable or non-biodegradable. Forexample, if the framework is non-biodegradable, the ECM can be removedby subjecting the framework to sonication, high pressure water jets,mechanical scraping, or mild treatment with detergents or enzymes, orany combination of the above.

If the framework is biodegradable, the ECM can be collected, forexample, by allowing the framework to degrade or dissolve in solution.Alternatively, if the biodegradable framework is composed of a materialthat can itself be injected along with the ECM, the framework and theECM can be processed in toto for subsequent injection. Alternatively,the ECM can be removed from the biodegradable framework by any of themethods described above for collection of ECM from a non-biodegradableframework. All collection processes are preferably designed so as not todenature the ECM produced by the cells of the invention.

Once the ECM has been collected, it may be processed further. The ECMcan be homogenized to fine particles using techniques well known in theart such as, for example, by sonication, so that they can pass through asurgical needle. ECM components can be crosslinked, if desired, by gammairradiation. Preferably, the ECM can be irradiated between 0.25 to 2mega rads to sterilize and crosslink the ECM. Chemical crosslinkingusing agents that are toxic, such as glutaraldehyde, is possible but notgenerally preferred.

Cell lysates prepared from the populations of the postpartum-derivedcells also have many utilities. In one embodiment, whole cell lysatesare prepared, e.g., by disrupting cells without subsequent separation ofcell fractions. In another embodiment, a cell membrane fraction isseparated from a soluble fraction of the cells by routine methods knownin the art, e.g., centrifugation, filtration, or similar methods. Use ofsoluble cell fractions in vivo allows the beneficial intracellularmilieu to be used in a patient without triggering rejection or anadverse response. Methods of lysing cells are well-known in the art andinclude various means of mechanical disruption, enzymatic disruption, orchemical disruption, or combinations thereof. Such cell lysates may beprepared from cells directly in their growth medium and thus containingsecreted growth factors and the like, or may be prepared from cellswashed free of medium in, for example, PBS or other solution. Washedcells may be resuspended at concentrations greater than the originalpopulation density if preferred. Cell lysates prepared from populationsof postpartum-derived cells may be used as is, further concentrated, byfor example, ultrafiltration or lyophilization, or even dried, partiallypurified, combined with pharmaceutically acceptable carriers or diluentsas are known in the art, or combined with other compounds such asbiologicals, for example pharmaceutically useful protein compositions.Cell lysates may be used in vitro or in vivo, alone or for example, withcells. The cell lysates, if introduced in vivo, may be introducedlocally at a site of treatment, or remotely to provide, for exampleneeded cellular growth factors to a patient.

The amounts and/or ratios of proteins may be adjusted by mixing the ECMor cell lysate produced by the cells of the invention with ECM or celllysate of one or more other cell types. In addition, biologically activesubstances such as proteins, growth factors and/or drugs, can beincorporated into the ECM or cell lysate preparation. Exemplarybiologically active substances include anti-inflammatory agents andgrowth factors which promote healing and tissue repair. Cells may beco-administered with the ECM or cell lysates of the invention. ECM orcell lysate of PDCs may be formulated for administration as describedabove for PDCs.

Use of PDCs for In Vitro Screening of Drug Efficacy or Toxicity

The cells and tissues of the invention may be used in vitro to screen awide variety of compounds for effectiveness and cytotoxicity ofpharmaceutical agents, growth/regulatory factors, anti-inflammatoryagents. To this end, the cells of the invention, or tissue culturesdescribed above, are maintained in vitro and exposed to the compound tobe tested. The activity of a cytotoxic compound can be measured by itsability to damage or kill cells in culture. This may readily be assessedby vital staining techniques. The effect of growth/regulatory factorsmay be assessed by analyzing the number of living cells in vitro, e.g.,by total cell counts, and differential cell counts. This may beaccomplished using standard cytological and/or histological techniques,including the use of immunocytochemical techniques employing antibodiesthat define type-specific cellular antigens. The effect of various drugson the cells of the invention either in suspension culture or in thethree-dimensional system described above may be assessed.

The cells and tissues of the invention may be used as model systems forthe study of physiological or pathological conditions. The cells andtissues of the invention may also be used to study the mechanism ofaction of cytokines, growth factors and inflammatory mediators, e.g.,IL-1, TNF and prostaglandins. In addition, cytotoxic and/orpharmaceutical agents can be screened for those that are mostefficacious for a particular patient. Agents that prove to beefficacious in vitro could then be used to treat the patienttherapeutically.

Use of PDCs to Produce Biological Molecules

In a further embodiment, the cells of the invention can be cultured invitro to produce biological products in high yield. For example, suchcells, which either naturally produce a particular biological product ofinterest (e.g., a growth factor, regulatory factor, or peptide hormone),or have been genetically engineered to produce a biological product,could be clonally expanded using, for example, the three-dimensionalculture system described above. If the cells excrete the biologicalproduct into the nutrient medium, the product can be readily isolatedfrom the spent or conditioned medium using standard separationtechniques, e.g., such as differential protein precipitation,ion-exchange chromatography, gel filtration chromatography,electrophoresis, and high performance liquid chromatography. A“bioreactor” may be used to take advantage of the flow method forfeeding, for example, a three-dimensional culture in vitro.

Essentially, as fresh media is passed through the three-dimensionalculture, the biological product is washed out of the culture and maythen be isolated from the outflow, as above.

Alternatively, a biological product of interest may remain within thecell and, thus, its collection may require that the cells are lysed. Thebiological product may then be purified using any one or more of theabove-listed techniques.

Kits

The PDCs and components and products thereof can conveniently beemployed as part of a kit, for example, for culture or implantation.Accordingly, the invention provides a kit including the PDCs andadditional components, such as a matrix (e.g., a scaffold), hydratingagents (e.g., physiologically-compatible saline solutions, prepared cellculture media), cell culture substrates (e.g., culture dishes, plates,vials, etc.), cell culture media (whether in liquid or powdered form),antibiotic compounds, hormones, and the like. While the kit can includeany such components, preferably it includes all ingredients necessaryfor its intended use. If desired, the kit also can include cells(typically cryopreserved), which can be seeded into the lattice asdescribed herein.

In another aspect, the invention provides kits that utilize the PDCs,PDC populations, components and products of PDCs in various methods foraugmentation, regeneration, and repair as described above. In someembodiments, the kits may include one or more cell populations,including at least PDCs and a pharmaceutically acceptable carrier(liquid, semi-solid or solid). The kits also optionally may include ameans of administering the cells, for example by injection. The kitsfurther may include instructions for use of the cells. Kits prepared forfield hospital use, such as for military use, may include full-proceduresupplies including tissue scaffolds, surgical sutures, and the like,where the cells are to be used in conjunction with repair of acuteinjuries. Kits for assays and in vitro methods as described herein maycontain one or more of (1) PDCs or components or products of PDCs, (2)reagents for practicing the in vitro method, (3) other cells or cellpopulations, as appropriate, and (4) instructions for conducting the invitro method.

Cryopreservation and Banking PDCs

PDCs of the invention can be cryopreserved and maintained or stored in a“cell bank”. Cryopreservation of cells of the invention may be carriedout according to known methods. For example, but not by way oflimitation, cells may be suspended in a “freeze medium” such as, forexample, culture medium further comprising 0 to 95 percent FBS and 0 to10 percent dimethylsulfoxide (DMSO), with or without 5 to 10 percentglycerol, at a density, for example, of about 0.5 to 10×10⁶ cells permilliliter. The cryopreservation medium may comprise cryopreservationagents including but not limited to methylcellulose. The cells aredispensed into glass or plastic ampoules that are then sealed andtransferred to the freezing chamber of a controlled rate freezer. Theoptimal rate of freezing may be determined empirically. A programmablerate freezer for example, can give a change in temperature of −1 to −10°C. per minute. The preferred cryopreservation temperature is about −80°C. to about −180° C., more preferably is about −90° C. to about −160°C., and most preferably is about −125 to about −140° C. Cryopreservedcells preferably are transferred to liquid nitrogen prior to thawing foruse. In some embodiments, for example, once the ampoules have reachedabout −90° C., they are transferred to a liquid nitrogen storage area.Cryopreserved cells can be stored for a period of years.

The cryopreserved cells of the invention constitute a bank of cells,portions of which can be “withdrawn” by thawing and then used as needed.Thawing should generally be carried out rapidly, for example, bytransferring an ampoule from liquid nitrogen to a 37° C. water bath. Thethawed contents of the ampoule should be immediately transferred understerile conditions to a culture vessel containing an appropriate mediumsuch as DMEM conditioned with 10 percent FBS.

In yet another aspect, the invention also provides for banking oftissues, cells, cellular components and cell populations of theinvention. As discussed above, the cells are readily cryopreserved. Theinvention therefore provides methods of cryopreserving the cells in abank, wherein the cells are stored frozen and associated with a completecharacterization of the cells based on immunological, biochemical andgenetic properties of the cells. The cells so frozen can be used forautologous, syngeneic, or allogeneic therapy, depending on therequirements of the procedure and the needs of the patient. Preferably,the information on each cryopreserved sample is stored in a computer,which is searchable based on the requirements of the surgeon, procedureand patient with suitable matches being made based on thecharacterization of the cells or populations. Preferably, the cells ofthe invention are grown and expanded to the desired quantity of cellsand therapeutic cell compositions are prepared either separately or asco-cultures, in the presence or absence of a matrix or support. Whilefor some applications it may be preferable to use cells freshlyprepared, the remainder can be cryopreserved and banked by freezing thecells and entering the information in the computer to associate thecomputer entry with the samples. Even where it is not necessary to matcha source or donor with a recipient of such cells, for immunologicalpurposes, the bank system makes it easy to match, for example, desirablebiochemical or genetic properties of the banked cells to the therapeuticneeds. Upon matching of the desired properties with a banked sample, thesample is retrieved, and readied for therapeutic use. Cell lysates orcomponents prepared as described herein may also be preserved (e.g.,cryopreserved, lyophilized) and banked in accordance with the presentinvention.

The following examples describe several aspects of embodiments of theinvention in greater detail. These examples are provided to furtherillustrate, not to limit, aspects of the invention described herein.

EXAMPLES Example 1 Derivation of Cells from Postpartum Placental Tissue

Postpartum placentas were obtained upon birth of either a full term orpre-term pregnancy. Cells were harvested from five separate donors ofplacental tissue. Different methods of cell isolation were tested fortheir ability to yield cells with: 1) the potential to differentiateinto cells with different phenotypes, or 2) the potential to providecritical trophic factors useful for other cells and tissues.

Methods & Materials

Isolation of Cells from Placenta.

Placental tissue was obtained from National Disease Resarch Interchange(NDRI) (Philadelphia, Pa.). The tissues were obtained from a pregnancyat the time of a normal surgical delivery. Placental cells were isolatedaseptically in a laminar flow hood. To remove blood and debris, thetissue was washed in phosphate buffered saline (PBS; Invitrogen,Carlsbad, Calif.) in the presence of 100 Units/milliliter penicillin,100 micrograms/milliliter streptomycin, and 0.25 micrograms/milliliteramphotericin (Invitrogen Carlsbad, Calif.). The tissues were thenmechanically dissociated in 150 cm² tissue culture plates in thepresence of 50 milliliters of medium (DMEM-Low glucose or DMEM-Highglucose; Invitrogen), until the tissue was minced into a fine pulp. Thechopped tissues were transferred to 50 milliliter conical tubes(approximately 5 grams of tissue per tube).

The tissue was then digested in either DMEM-Low glucose medium orDMEM-High glucose medium, each containing 100 Units/milliliterpenicillin, 100 micrograms/milliliter streptomycin, and 0.25micrograms/milliliter amphotericin and digestion enzymes. In someexperiments an enzyme mixture of collagenase and dispase was used(“C:D;” collagenase (Sigma, St Louis, Mo.), 500 Units/milliliter; anddispase (Invitrogen), 50 Units/milliliter in DMEM-Low glucose medium).In other experiments a mixture of collagenase, dispase and hyaluronidase(“C:D:H”) was used (collagenase, 500 Units/milliliter; dispase, 50Units/milliliter; and hyaluronidase (Sigma), 5 Units/milliliter, inDMEM-Low glucose). The conical tubes containing the tissue, medium anddigestion enzymes were incubated at 37° C. in an orbital shaker(Environ, Brooklyn, N.Y.) at 225 rpm for 2 hours.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,and the supernatant was aspirated. The pellet was resuspended in 20milliliter of Growth medium (DMEM:Low glucose (Invitrogen), 15 percent(v/v) fetal bovine serum (FBS; defined bovine serum; Lot#AND18475;Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol (Sigma), 100Units/milliliter penicillin, 100 micrograms/milliliter streptomycin,0.25 micrograms/milliliter amphotericin B; Invitrogen, Carlsbad,Calif.)). The cell suspension was filtered through a 70-micrometer nyloncell strainer (BD Biosciences). An additional 5 milliliters rinsecomprising Growth medium was passed through the strainer. The cellsuspension was then passed through a 40-micrometer nylon cell strainer(BD Biosciences) and chased with a rinse of an additional 5 millilitersof Growth medium.

The filtrate was resuspended in Growth medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cells were resuspended in 50 milliliters of freshgrowth medium. This process (i.e., resuspension, centrifugation, andaspiration) was repeated twice more.

After the final centrifugation, supernatant was aspirated and the cellpellet was resuspended in 5 milliliters of fresh growth medium. Thenumber of viable cells was determined using Trypan Blue staining. Cellswere then cultured under standard conditions.

The cells isolated from placenta were seeded at 5,000 cells/cm² ontogelatin-coated T-75 cm² flasks (Corning Inc., Corning, N.Y.) in Growthmedium (DMEM-Low glucose (Invitrogen), 15 percent (v/v) defined bovineserum (Hyclone, Logan, Utah; Lot#AND18475), 0.001 percent (v/v)2-mercaptoethanol (Sigma), 100 Units/milliliter penicillin, 100micrograms/milliliter streptomycin, and 0.25 micrograms/milliliteramphotericin (Invitrogen)). After about 2-4 days, spent medium wasaspirated from the flasks. Cells were washed with PBS three times toremove debris and blood-derived cells. Cells were then replenished withGrowth medium and allowed to grow to confluence (about 10 days frompassage 0 to passage 1). On subsequent passages (from passage 1 to 2,etc.), cells reached sub-confluence (75-85 percent confluence) in 4-5days. For these subsequent passages, cells were seeded at 5,000cells/cm². Cells were grown in a humidified incubator with 5 percentcarbon dioxide and 20 percent oxygen, at 37° C.

Isolation of Populations of Maternal-Derived and Neonatal-Derived Cellsfrom Placenta.

The cell isolation protocol was performed aseptically in a laminar flowhood. The placental tissue was washed in phosphate buffered saline (PBS;Invitrogen, Carlsbad, Calif.) in the presence of antimycotic andantibiotic (100 Units/milliliter penicillin, 100 microgram/milliliterstreptomycin, 0.25 microgram/milliliter amphotericin B; Invitrogen) toremove blood and debris. The placental tissue was then dissected intothree sections: top-line (neonatal side or aspect), mid-line (mixed cellisolation neonatal and maternal, or villous region), and bottom line(maternal side or aspect).

The separated sections were individually washed several times in PBSwith antibiotic/antimycotic to further remove blood and debris. Eachsection was then mechanically dissociated in 150 cm² tissue cultureplates in the presence of 50 milliliters of DMEM-Low glucose(Invitrogen) to a fine pulp. The pulp was transferred to 50 milliliterconical tubes. Each tube contained approximately 5 grams of tissue. Thetissue was digested in either DMEM-Low glucose or DMEM-High glucosemedium containing 100 Units/milliliter penicillin, 100micrograms/milliliter streptomycin, and 0.25 micrograms/milliliteramphotericin and digestion enzymes. In some experiments an enzymemixture of collagenase and dispase (“C:D”) was used containingcollagenase (Sigma, St Louis, Mo.) at 500 Units/milliliter and dispase(Invitrogen) at 50 Units/milliliter in DMEM-Low glucose medium. In otherexperiments a mixture of collagenase, dispase, and hyaluronidase(“C:D:H”) was used (collagenase, 500 Units/milliliter; dispase, 50Units/milliliter; and hyaluronidase (Sigma), 5 Units/milliliter inDMEM-Low glucose). The conical tubes containing the tissue, medium, anddigestion enzymes were incubated for 2 hours at 37° C. in an orbitalshaker (Environ, Brooklyn, N.Y.) at 225 rpm.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,and the resultant supernatant was aspirated off. The pellet wasresuspended in 20 milliliters of Growth medium (DMEM-Low glucose(Invitrogen), 15% (v/v) fetal bovine serum (FBS; defined bovine serum;Lot#AND18475; Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol(Sigma, St. Louis, Mo.), 100 Units/milliliter penicillin, 100microgram/milliliter streptomycin, 0.25 microgram/milliliteramphotericin B; Invitrogen)). The cell suspension was filtered through a70 micrometer nylon cell strainer (BD Biosciences), chased by a rinsewith an additional 5 milliliters of Growth medium. The total cellsuspension was passed through a 40 micrometer nylon cell strainer (BDBiosciences) followed with an additional 5 milliliters of Growth mediumas a rinse.

The filtrate was resuspended in Growth medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated, and the cell pellet was resuspended in 50 milliliters offresh Growth medium. This process (i.e., resuspension, centrifugation,and aspiration) was repeated twice more.

After the final centrifugation, supernatant was aspirated, and the cellpellet was resuspended in 5 milliliters of fresh Growth medium. A cellcount was determined using the Trypan Blue Exclusion test. Cells werethen cultured at standard conditions.

Isolation of PDCs Using Different Growth Conditions.

Placenta-derived cells were digested in growth medium with or without0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.), using the enzymecombination of C:D:H, according to the procedures provided above.Placenta-derived cells so isolated were seeded under conditions setforth in Table 1-1 and grown in the presence of penicillin/streptomycin.Cells were passaged up to four times after seeding and cryopreserved.The cryopreserved cells were banked.

TABLE 1-1 Isolation and culture expansion of placental cells undervarying conditions: Condi- Growth tion Medium FBS BME Gelatin O₂ Factors1 DMEM-Lg 15% Y Y 20% N 2 DMEM-Lg 15% Y Y  5% N 3 DMEM-Lg 15% Y N 20% N4 DMEM-Lg 15% Y N  5% N 5 DMEM-Lg  2% Y N 20% EGF/FGF (Laminin) (20ng/ml) 6 DMEM-Lg  2% Y N  5% EGF/FGF (Laminin) (20 ng/ml) 7 DMEM-Lg  2%Y N 20% PDGF/ (Fibronectin) VEGF 8 DMEM-Lg  2% Y N  5% PDGF/(Fibronectin) VEGF 9 DMEM-Lg 15% N Y 20% N 10 DMEM-Lg 15% N Y  5% N 11DMEM-Lg 15% N N 20% N 12 DMEM-Lg 15% N N  5% N 13 DMEM-Lg  2% N N 20%EGF/FGF (Laminin) (20 ng/ml) 14 DMEM-Lg  2% N N  5% EGF/FGF (Laminin)(20 ng/ml) 15 DMEM-Lg  2% N N 20% PDGF/ (Fibronectin) VEGF 16 DMEM-Lg 2% N N  5% PDGF/ (Fibronectin) VEGF Key: Lg: Low glucose; N: none; Y:yes; BME: beta-mercaptoethanol; FGF: fibroblast growth factor; EGF:epidermal growth factor; PDGF: platelet-derived growth factor; VEGF:vascular endothelial growth factor.

Results

Isolation of PDCs Using Different Growth Conditions.

In all conditions set forth in Table 1-1, cells attached and expandedwell between passage 0 and 1. Cells in condition 5 to 8 and 13 to 16were demonstrated to proliferate well up to at least four passages afterseeding.

Isolation of Cells from Placenta Using Different Enzyme Combinations.

Tissue digestion with collagenase: dispase andcollagenase:dispase:hyaluronidase resulted in the isolation of cellpopulations from placental tissues that expanded readily.

Summary.

PDCs can be isolated using a combination of a matrix metalloprotease andneutral protease, such as but not limited to a combination ofcollagenase and dispase. PDCs are preferably isolated using an enzymecombination of a matrix metalloprotease, a neutral protease, and amucolytic enzyme that degrades hyaluronic acid, such as but not limitedto a combination of collagenase, dispase, and hyaluronidase or acombination of LIBERASE (Boehringer Mannheim Corp., Indianapolis, Ind.)and hyaluronidase. Blendzyme 3, which is collagenase (4 Wunsch units/g)and thermolysin (1714 casein Units/g) may be used together withhyaluronidase to isolate cells.

Example 2 Evaluation of Growth Media for Placenta-Derived Cells

Several cell culture media were evaluated for their ability to supportthe growth of placenta-derived cells. The growth of placenta-derivedcells in normal (20%) and low (5%) oxygen was assessed after 3 daysusing the MTS colorimetric assay.

Methods & Materials

Placenta-derived cells at passage 8 (P8) were seeded at 1×10³ cells/wellin 96 well plates in Growth medium (DMEM-low glucose (Gibco, CarlsbadCalif.), 15% (v/v) fetal bovine serum (Cat. #SH30070.03; Hyclone, Logan,Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, Mo.), 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin(Gibco). After 8 hours the medium was changed to that described in Table2-1 and cells were incubated in normal (20%, v/v) or low (5%, v/v)oxygen at 37° C., 5% CO₂ for 48 hours. MTS was added to the culturemedium (CELLTITER96 AQueous One Solution Cell Proliferation Assay,Promega, Madison, Wis.) for 3 hours and the absorbance measured at 490nanometer (Molecular Devices, Sunnyvale Calif.).

TABLE 2-1 Culture media evaluated Added fetal bovine serum CultureMedium Supplier % (v/v) DMEM-low glucose Gibco Carlsbad CA 0, 2, or 10DMEM-high glucose Gibco Carlsbad CA 0, 2, or 10 RPMI 1640 Mediatech,Inc. 0, 2, or 10 Herndon, VA Cell gro-free Mediatech, Inc. —(Serum-free, Protein-free) Herndon, VA Ham's F10 Mediatech, Inc. 0, 2,or 10 Herndon, VA MSCGM (complete with Cambrex, 0, 2, or 10 serum)Walkersville, MD Complete-serum free Mediatech, Inc. — w/albuminHerndon, VA Growth medium NA — Ham's F12 Mediatech, Inc. 0, 2, or 10Herndon, VA Iscove's Mediatech, Inc. 0, 2, or 10 Herndon, VA BasalMedium Eagle's Mediatech, Inc. 0, 2, or 10 Herndon, VA DMEM/F12 (1:1)Mediatech, Inc. 0, 2, or 10 Herndon, VA MSCGM: Mesenchymal Stem CellGrowth Medium

Results

Standard curves for the MTS assay established a linear correlationbetween an increase in absorbance and an increase in cell number. Theabsorbance values obtained were converted into estimated cell numbersand the change (%) relative to the initial seeding was calculated.

The addition of serum to media at normal oxygen conditions resulted in areproducible dose-dependent increase in absorbance and thus the viablecell number (extrapolated). The addition of serum to complete MSCGMresulted in a dose-dependent decrease in absorbance. In the mediawithout added serum, cells only grew in Cellgro, Ham's F10, and DMEM.

Reduced oxygen increased the growth rate of cells in Growth medium,Ham's F10, and, MSCGM.

In decreasing order of growth, the media resulting in the best growth ofthe PDCs were Growth medium>MSCGM>Iscove's+10% FBS=DMEM-High glucose+10%FBS=Ham's F12+10% FBS=RPMI 1640+10% FBS.

Summary.

Placenta-derived cells may be grown in a variety of culture media innormal or low oxygen. PDCs grew in serum-free conditions, for example,in Ham's F10, Cellgro-free, and DMEM. PDCs also grew in protein-freeconditions, for example, in Ham's F10 and Cellgro-free. Reduced oxygenincreased the growth rate of cells in Growth medium, Ham's F10, and,MSCGM.

REFERENCE

-   U.S. Application Publication No. 20040005704

Example 3 Growth of Postpartum Cells in Medium Containing D-Valine

It has been reported that medium containing D-valine instead of thenormal L-valine isoform can be used to selectively inhibit the growth offibroblast-like cells in culture (Hongpaisan (2000) Cell Biol Int.24:1-7; Sordillo et al. (1988) Cell Biol Int Rep. 0.12:355-64).Experiments were performed to determine whether placenta-derived cellscould grow in medium containing D-valine.

Methods & Materials

Placenta-derived cells (P3) and fibroblasts (P9) were seeded at 5×10³cells/cm² in gelatin-coated T75 flasks (Corning, Corning, N.Y.). After24 hours the medium was removed and the cells were washed with phosphatebuffered saline (PBS) (Gibco, Carlsbad, Calif.) to remove residualmedium. The medium was replaced with a Modified Growth medium (DMEM withD-valine (special order, Gibco), 15% (v/v) dialyzed fetal bovine serum(Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma), 50Units/milliliter penicillin, 50 micrograms/milliliter streptomycin(Gibco)).

Results

Placenta-derived and fibroblast cells seeded in the D-valine-containingmedium did not proliferate, unlike cells seeded in growth mediumcontaining dialyzed serum. Fibroblast cells changed morphologically,increasing in size and changing shape. All of the cells died andeventually detached from the flask surface after 4 weeks.

Summary.

PDCs require L-valine for cell growth and to maintain long-termviability.

Example 4 Cryopreservation Media for Placenta-Derived Cells

The objective of this study was to determine a suitable cryopreservationmedium for the cryopreservation of placenta-derived cells.

Methods & Materials

Placenta-derived cells grown in Growth medium (DMEM-low glucose (Gibco,Carlsbad Calif.), 15% (v/v) fetal bovine serum (Cat. #SH30070.03,Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St.Louis, Mo.), 50 Units/milliliter penicillin, 50 microgram/milliliterstreptomycin (Gibco)), in a gelatin-coated T75 flask were washed withphosphate buffered saline (PBS; Gibco) and trypsinized using 1milliliter Trypsin/EDTA (Gibco). The trypsinization was stopped byadding 10 milliliters Growth medium. The cells were centrifuged at150×g, supernatant removed, and the cell pellet was resuspended in 1milliliter Growth medium. An aliquot of cell suspension, 60 microliter,was removed and added to 60 microliter □trypan blue (Sigma). The viablecell number was estimated using a hemocytometer. The cell suspension wasdivided into four equal aliquots each containing 88×10⁴ cells each. Thecell suspension was centrifuged and resuspended in 1 milliliter of eachmedia below and transferred into Cryovials (Nalgene).

1.) Growth medium +10% (v/v) DMSO (Hybrimax, Sigma, St. Louis, Mo.)

2.) Cell Freezing medium w/DMSO, w/methylcellulose, serum-free (C6295,Sigma, St. Louis, Mo.)

3.) Cell Freezing medium serum-free (C2639, Sigma, St. Louis, Mo.)

4.) Cell Freezing Medium w/glycerol (C6039, Sigma, St. Louis, Mo.)

The cells were cooled at approximately 1° C./min overnight in a −80° C.freezer using a “Mr Frosty” freezing container according to themanufacturer's instructions (Nalgene, Rochester, N.Y.). Vials of cellswere transferred into liquid nitrogen for 2 days before thawing rapidlyin a 37° C. water bath. The cells were added to 10 milliliters Growthmedium and centrifuged before the cell number and viability wasestimated as before. Cells were seeded onto gelatin-coated flasks at5,000 cells/cm² to determine whether the cells would attach andproliferate.

Results

The initial viability of the cells to be cryopreserved was assessed bytrypan blue staining to be 100%.

There was a commensurate reduction in cell number with viability forC6295 due to cell lysis. The viable cells cryopreserved in all foursolutions attached, divided, and produced a confluent monolayer within 3days. There was no discernable difference in estimated growth rate.

Summary.

The cryopreservation of cells is one procedure available for preparationof a cell bank or a cell product. Four cryopreservation mixtures werecompared for their ability to protect human placenta-derived cells fromfreezing damage. Dulbecco's modified Eagle's medium (DMEM) and 10% (v/v)dimethylsulfoxide (DMSO) is a preferred medium of those compared forcryopreservation of placenta-derived cells.

Example 5 Growth Characteristics of Placenta-Derived Cells

The cell expansion potential of placenta-derived cells was compared toother populations of isolated stem cells. The process of cell expansionto senescence is referred to as Hayflick's limit (Hayflick (1974) J. Am.Geriatr. Soc. 22:1-12; Hayflick (1974) Gerontologist 14:37-45).

Materials and Methods

Gelatin-coating flasks. Tissue culture plastic flasks were coated byadding 20 milliliters 2% (w/v) porcine gelatin (Type B: 225 Bloom;Sigma, St Louis, Mo.) to a T75 flask (Corning, Corning, N.Y.) for 20minutes at room temperature. After removing the gelatin solution, 10milliliters phosphate-buffered saline (PBS) (Invitrogen, Carlsbad,Calif.) were added and then aspirated.

Comparison of Expansion Potential of Placenta-Derived Cells with OtherCell Populations.

For comparison of growth expansion potential, the following cellpopulations were utilized: i) Mesenchymal stem cells (MSC; Cambrex,Walkersville, Md.); ii) Adipose-derived cells (U.S. Pat. No. 6,555,374B1; U.S. Patent Application US20040058412); iii) Normal dermal skinfibroblasts (cc-2509 lot #9F0844; Cambrex, Walkersville, Md.); and iv)Placenta-derived cells. Cells were initially seeded at 5,000 cells/cm²on gelatin-coated T75 flasks in Growth medium (DMEM-Low glucose(Invitrogen, Carlsbad, Calif.), 15% (v/v) defined bovine serum (Hyclone,Logan, Utah; Lot#AND18475), 0.001% (v/v) 2-mercaptoethanol (Sigma, St.Louis, Mo.), 100 Units/milliliter penicillin, 100 microgram/milliliterstreptomycin, 0.25 micrograms/milliliter amphotericin B; Invitrogen,Carlsbad, Calif.)). For subsequent passages, cell cultures were treatedas follows. After trypsinization, viable cells were counted after TrypanBlue staining (e.g., cell suspension (50 microliters) was combined withTrypan Blue (50 microliters, Sigma, St. Louis Mo.); viable cell numberswere estimated using a hemocytometer).

Following counting, cells were seeded at 5,000 cells/cm² ontogelatin-coated T 75 flasks in 25 milliliters of fresh growth medium.Cells were grown under standard atmosphere with 5% carbon dioxide at 37°C. The growth medium was changed twice per week. When cells reachedabout 85 percent confluence, they were passaged. This process wasrepeated until the cells reached senescence.

At each passage, cells were trypsinized and counted. The viable cellyield, population doubling [ln (cell final/cell initial)/ln 2], anddoubling time (time in culture (h)/population doubling) were calculated.For the purposes of determining optimal cell expansion, the total cellyield per passage was determined by multiplying the total yield for theprevious passage by the expansion factor for each passage (i.e.,expansion factor=cell final/cell initial).

Expansion Potential of Cell Banks at Low Density.

The expansion potential of cells banked at passage 10 was tested. Normaldermal skin fibroblasts (cc-2509 lot #9F0844; Cambrex, Walkersville,Md.) and placenta-derived cells were tested. These cell populations hadbeen banked at passage 10 previously, having been seeded at 5,000cell/cm² and grown to confluence at each passage to that point. Theeffect of cell density on the cell populations following cell thaw atpassage 10 was determined. Cells were thawed under standard conditionsand counted using Trypan Blue staining. Thawed cells were then seeded at1,000 cells/cm² in Growth medium (DMEM:Low glucose (Invitrogen,Carlsbad, Calif.), 15% (v/v) defined bovine serum (Hyclone, Logan, Utah;Lot#AND18475), 0.001% 2-mercaptoethanol (Sigma, St. Louis, Mo.), 100Units/milliliter penicillin, 100 micrograms/milliliter streptomycin, and0.25 micrograms/milliliter amphotericin (Invitrogen, Carlsbad, Calif.)).Cells were grown under standard atmospheric conditions at 37° C. Growthmedium was changed twice a week and cells were passaged as they reachedabout 85% confluence. Cells were subsequently passaged until senescence.Cells were trypsinized and counted at each passage. The cell yield,population doubling (ln (cell final/cell initial)/ln2), and doublingtime (time in culture (h)/population doubling) were calculated for eachpassage. The total cell yield per passage was determined by multiplyingtotal yield for the previous passage by the expansion factor for eachpassage (i.e., expansion factor=cell final/cell initial).

Expansion of Placenta-Derived Cells at Low Density from Initial CellSeeding.

The expansion potential of freshly isolated placenta-derived cellcultures under low cell seeding conditions was tested in anotherexperiment. Placenta-derived cells were isolated as described inExample 1. Cells were seeded at 1,000 cells/cm² and passaged asdescribed above until senescence. Cells were grown under standardatmospheric conditions at 37° C. Growth medium was changed twice perweek. Cells were passaged as they reached about 85% confluence. At eachpassage, cells were trypsinized and counted by Trypan Blue staining. Thecell yield, population doubling (ln (cell final/cell initial)/ln 2) anddoubling time (time in culture (h)/population doubling) were calculatedfor each passage. The total cell yield per passage was determined bymultiplying the total yield for the previous passage by the expansionfactor for each passage (i.e., expansion factor=cell final/cellinitial). Cells were grown on gelatin and non-gelatin coated flasks.

Expansion of Clonal Neonatal or Maternal Placenta-Derived Cells.

Cloning may be used in order to expand a population of neonatal ormaternal cells successfully from placental tissue. Following isolationof three different cell populations from the placenta (neonatal aspect,maternal aspect, and villous region), these cell populations areexpanded under standard growth conditions and then karyotyped to revealthe identity of the isolated cell populations. By isolating the cellsfrom a mother who delivers a boy, it is possible to distinguish betweenthe male and female chromosomes by performing metaphase spreads. Theseexperiments can be used to demonstrate that top-line cells are karyotypepositive for neonatal phenotype, mid-line cells are karyotype positivefor both neonatal and maternal phenotypes, and bottom-line cells arekaryotype positive for maternal cells.

Other Growth Conditions.

In other experiments cells were expanded on either non-coated,collagen-coated, fibronectin-coated, laminin-coated, or extracellularmembrane protein (e.g., MATRIGEL (BD Discovery Labware, Bedford,Mass.))-coated plates. Cultures have been demonstrated to expand well onthese different matrices.

Results

Comparison of Expansion Potential of Placenta-Derived Cells with OtherCell Populations.

Placenta-derived cells expanded for greater than 40 passages, generatingcell yields of >1×10¹⁷ cells in 60 days. In contrast, MSCs andfibroblasts senesced after <25 days and <60 days, respectively. Althoughboth adipose-derived and omental cells expanded for almost 60 days, theygenerated total cell yields of 4.5×10¹² and 4.24×10¹³, respectively.Thus, when seeded at 5,000 cells/cm² under the experimental conditionsutilized, PDCs expanded much better than the other cell types grownunder the same conditions (Table 5-1).

TABLE 5-1 Growth characteristics for different cell populations grown tosenescence Total Population Total Cell Cell Type Senescence DoublingsYield MSC 24 day 8 4.72 × 10⁷  Adipose-derived cells 57 day 24  4.5 ×10¹² (Artecel, U.S. Pat. No. 6,555,374) Fibroblasts 53 day 26 2.82 ×10¹³ Placenta 80 day 46 2.49 × 10¹⁹

Expansion of Potential of Cell Banks at Low Density.

Placenta-derived and fibroblast cells expanded for greater than 10passages generating cell yields of >1×10¹¹ cells in 60 days (Table 5-2).After 60 days under these conditions, the placenta-derived cells becamesenescent whereas the fibroblast cell populations senesced after 80days, completing >40 population doublings.

TABLE 5-2 Growth characteristics for different cell populations usinglow density growth expansion from passage 10 through senescence CellType Total Population Total Cell (Passage No.) Senescence DoublingsYield Fibroblast (P10) 80 day 43.68 2.59 × 10¹¹ Placental (P10) 60 day32.96 6.09 × 10¹²

Expansion of Placenta-Derived Cells at Low Density from Initial CellSeeding.

Placenta-derived cells were expanded at low density (1,000 cells/cm²) ongelatin-coated and uncoated plates or flasks. Growth potential of thesecells under these conditions was good. The cells expanded readily in alog phase growth. The rate of cell expansion was similar to thatobserved when placenta-derived cells were seeded at 5,000 cells/cm² ongelatin-coated flasks in growth medium. No differences were observed incell expansion potential between culturing on either uncoated flasks orgelatin-coated flasks. Cells grown in gelatin-coated flasks appearedphenotypically smaller than cells grown in uncoated flasks.

Expansion of Clonal Neonatal or Maternal Placenta-Derived Cells.

A clonal neonatal or maternal cell population can be expanded fromplacenta-derived cells isolated from the neonatal aspect or the maternalaspect, respectively, of the placenta. Cells are serially diluted andthen seeded onto gelatin-coated plates in Growth medium for expansion at1 cell/well in 96-well gelatin coated plates. From this initial cloning,expansive clones are identified, trypsinized, and reseeded in 12-wellgelatin-coated plates in Growth medium and then subsequently passagedinto T25 gelatin-coated flasks at 5,000 cells/cm² in Growth medium.Subcloning is performed to ensure that a clonal population of cells hasbeen identified. For subcloning experiments, cells are trypsinized andreseeded at 0.5 cells/well. The subclones that grow well are expanded ingelatin-coated T25 flasks at 5,000 cells cm²/flask. Cells are passagedat 5,000 cells cm²/T75 flask. The growth characteristics of the bestclone are plotted, to demonstrate cell expansion. Karyotyping analysiscan confirm that the clone is either neonatal or maternal.

Summary.

The current cell expansion conditions of growing isolated PDCs atdensities of about 5,000 cells/cm² in growth medium on gelatin-coated oruncoated flasks under standard atmospheric oxygen are sufficient togenerate large numbers of cells at passage 11. PDCs also can be readilyexpanded using lower density culture conditions (e.g., about 1,000cells/cm²). It is preferred to culture placenta-derived cells understandard atmospheric conditions to generate large pools of cells.Culture conditions may be altered to achieve alternative proliferativeand/or differentiative capacity of placenta-derived cells.

Under the conditions utilized, while the expansion potential of MSCs andadipose-derived cells was limited, placenta-derived cells expand readilyto large numbers. The data demonstrate that placenta-derived cell linesas developed herein can expand for greater than 40 doublings to providesufficient cell numbers, for example, for cell banks, whereasmesenchymal stem cells cannot be expanded to obtain large quantities ofcells.

REFERENCES

-   Hayflick (1974) J. Am. Geriatr. Soc. 22(1):1-12

Example 6 Karyotype Analysis of Placenta-Derived Cells

Cell lines used in cell therapy are preferably homogeneous and free fromany contaminating cell type. Human cells used in cell therapy shouldhave a normal chromosome number (46) and structure. To identifyplacenta-derived cell lines that are homogeneous and free from cells ofnon-placental tissue origin, karyotypes of cell samples were analyzed.

Materials and Methods

PDCs from postpartum tissue of a male neonate were cultured in Growthmedium (DMEM-low glucose (Gibco Carlsbad, Calif.), 15% (v/v) fetalbovine serum (FBS) (Hyclone, Logan, Utah), 0.001% (v/v)betamercaptoethanol (Sigma, St. Louis, Mo.), and 50 Units/milliliterpenicillin, 50 microgram/milliliter streptomycin (Gibco, Carlsbad,Calif.)). Postpartum tissue from a male neonate (X,Y) was selected toallow distinction between neonatal-derived cells and maternal-derivedcells (X,X). Cells were seeded at 5,000 cells/cm² in Growth medium in aT25 flask (Corning, Corning, N.Y.) and expanded to about 80% confluence.A T25 flask containing cells was filled to the neck with Growth medium.Samples were delivered to a clinical cytogenetics lab by courier(estimated lab to lab transport time is one hour). Chromosome analysiswas performed by the Center for Human & Molecular Genetics at the NewJersey Medical School, Newark, N.J. Cells were analyzed during metaphasewhen the chromosomes are best visualized. Of twenty cells in metaphasecounted, five were analyzed for normal homogeneous karyotype number(two). A cell sample was characterized as homogeneous if two karyotypeswere observed. A cell sample was characterized as heterogeneous if morethan two karyotypes were observed. Additional metaphase cells werecounted and analyzed when a heterogeneous karyotype number (four) wasidentified.

Results

All cell samples sent for chromosome analysis were interpreted asexhibiting a normal appearance. Three of the thirteen cell linesanalyzed exhibited a heterogeneous phenotype (XX and XY) indicating thepresence of cells derived from both neonatal and maternal origins (Table6-1). Cells derived from tissue Placenta-N were isolated from theneonatal aspect of placenta. At passage zero, this cell line appearedhomogeneous XY. However, at passage nine, the cell line washeterogeneous (XX/XY), indicating a previously undetected presence ofcells of maternal origin.

TABLE 6-1 Karyotype analysis of PDCs Metaphase Metaphase cells cellsNumber of ISCN Tissue passage counted analyzed karyotypes KaryotypePlacenta 22 20 5 2 46, XX Placenta 2 20 5 2 46, XX Placenta-N 0 20 5 246, XY Placenta-V 0 20 5 2 46, XY Placenta-M 0 21 5 4 46, XY[18]/ 46,XX[3] Placenta-M 4 20 5 2 46, XX Placenta-N 9 25 5 4 46, XY[5]/ 46,XX[20] Placenta-N 1 20 5 2 46, XY C1 Placenta-N 1 20 6 4 46, XY[2]/ C346, XX[18] Placenta-N 1 20 5 2 46, XY C4 Placenta-N 1 20 5 2 46, XY C15Placenta-N 1 20 5 2 46, XY C20 Placenta-N 1 20 5 2 46, XY C22 Key:N—Neonatal side; V—villous region; M—maternal side; C—clone

Summary.

Chromosome analysis identified placenta-derived cells whose karyotypesappear normal as interpreted by a clinical cytogenetic laboratory.Karyotype analysis also identified cell lines free from maternal cells,as determined by homogeneous karyotype.

Example 7 Evaluation of Human Placenta-Derived Cell Surface Markers byFlow Cytometry

Characterization of cell surface proteins or “markers” by flow cytometrycan be used to determine a cell line's identity. The consistency ofexpression can be determined from multiple donors and in cells exposedto different processing and culturing conditions. Postpartum cell linesderived from the placenta were characterized (by flow cytometry)providing a profile for the identification of these cell lines.

Materials and Methods

Media.

Cells were cultured in Growth medium (DMEM-low glucose (Gibco Carlsbad,Calif.), 15% (v/v) fetal bovine serum (FBS); (Hylcone, Logan, Utah),0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, Mo.), and 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin(Gibco, Carlsbad, Calif.)).

Culture Vessels.

Cells were cultured in plasma-treated T75, T150, and T225 tissue cultureflasks (Corning, Corning, N.Y.) until confluent. The growth surfaces ofthe flasks were coated with gelatin by incubating 2% (w/v) gelatin(Sigma, St. Louis, Mo.) for 20 minutes at room temperature.

Antibody Staining.

Adherent cells in flasks were washed in phosphate buffered saline (PBS);(Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco,Carlsbad, Calif.). Cells were harvested, centrifuged, and resuspended in3% (v/v) FBS in PBS at a cell concentration of 1×10⁷ per milliliter. Inaccordance with the manufacturer's specifications, antibody to the cellsurface marker of interest (Table 7-1) was added to one hundredmicroliters of cell suspension, and the mixture was incubated in thedark for 30 minutes at 4° C. After incubation, cells were washed withPBS and centrifuged to remove unbound antibody. Cells were resuspendedin 500 microliters PBS and analyzed by flow cytometry.

Flow Cytometry Analysis.

Flow cytometry analysis was performed with a FACScalibur instrument(Becton Dickinson, San Jose, Calif.).

Antibodies to Cell Surface Markers.

The following antibodies to cell surface markers were used.

TABLE 7-1 Antibodies to cell surface markers Catalog AntibodyManufacture Number CD10 BD Pharmingen (San Diego, CA) 555375 CD13 BDPharmingen (San Diego, CA) 555394 CD31 BD Pharmingen (San Diego, CA)555446 CD34 BD Pharmingen (San Diego, CA) 555821 CD44 BD Pharmingen (SanDiego, CA) 555478 CD45RA BD Pharmingen (San Diego, CA) 555489 CD73 BDPharmingen (San Diego, CA) 550257 CD90 BD Pharmingen (San Diego, CA)555596 CD117 BD Biosciences (San Jose, CA) 340529 CD141 BD Pharmingen(San Diego, CA) 559781 PDGFr-alpha BD Pharmingen (San Diego, CA) 556002HLA-A, B, C BD Pharmingen (San Diego, CA) 555553 HLA-DR, DP, DQ BDPharmingen (San Diego, CA) 555558 IgG-FITC Sigma (St. Louis, MO) F-6522IgG- PE Sigma (St. Louis, MO) P-4685

Passage to Passage Comparison.

Placenta-derived cells were analyzed at passages 8, 15, and 20.

Donor to Donor Comparison.

To compare differences among donors, placenta cells from differentdonors were compared to each other.

Surface Coating Comparison.

Placenta-derived cells cultured on gelatin-coated flasks was compared toplacenta-derived cells cultured on uncoated flasks.

Digestion Enzyme Comparison.

Four treatments used for isolation and preparation of cells werecompared. Cells isolated from placenta by treatment with 1) collagenase;2) collagenase/dispase; 3) collagenase/hyaluronidase; and 4)collagenase/hyaluronidase/dispase were compared.

Placental Layer Comparison.

Cells isolated from the maternal aspect of placental tissue werecompared to cells isolated from the villous region of placental tissueand cells isolated from the neonatal fetal aspect of placenta.

Results

Placenta-Derived Cell Characterization.

Placenta-derived cells analyzed by flow cytometry showed positive forproduction of CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B,C, indicated by the increased values of fluorescence relative to the IgGcontrol. These cells were negative for detectable for production ofCD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, indicated byfluorescence values comparable to the IgG control. Variations influorescence values of positive curves were accounted. While the mean(i.e., CD13) and range (i.e., CD90) of the positive curves showed somevariation, the curves appeared normal, confirming a homogeneouspopulation, and exhibited fluorescence values greater than the IgGcontrol.

Passage to Passage Comparison.

Placenta-derived cells at passages 8, 15, and 20 analyzed by flowcytometry were positive for production of CD10, CD13, CD44, CD73, CD 90,PDGFr-alpha, and HLA-A, B, C, as reflected in the increased value offluorescence relative to the IgG control. The cells were negative forproduction of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, asindicated by fluorescence values consistent with the IgG control.

Donor to Donor Comparison.

Placenta-derived cells isolated from separate donors analyzed by flowcytometry each expressed CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha, andHLA-A, B, C, with increased values of fluorescence relative to the IgGcontrol. The cells were negative for production of CD31, CD34, CD45,CD117, CD141, and HLA-DR, DP, DQ as indicated by fluorescence valueconsistent with the IgG control.

The Effect of Surface Coating with Gelatin.

Placenta-derived cells expanded on either gelatin-coated or uncoatedflasks analyzed by flow cytometry expressed of CD10, CD13, CD44, CD73,CD 90, PDGFr-alpha, and HLA-A, B, C, reflected in the increased valuesof fluorescence relative to the IgG control. These cells were negativefor production of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQindicated by fluorescence values consistent with the IgG control.

Effect of Enzyme Digestion Procedure on the Cell Surface Marker Profile.

PDCs isolated using various digestion enzymes analyzed by flow cytometryexpressed CD10, CD13, CD44, CD73, CD 90, PDGFr-alpha, and HLA-A, B, C,as indicated by the increased values of fluorescence relative to the IgGcontrol. These cells were negative for production of CD31, CD34, CD45,CD117, CD141, and HLA-DR, DP, DQ, as indicated by fluorescence valuesconsistent with the IgG control.

Placental Layer Comparison.

Cells isolated from the maternal, villous, and neonatal layers of theplacenta, respectively, analyzed by flow cytometry showed positive forproduction of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, and HLA-A, B,C, as indicated by the increased value of fluorescence relative to theIgG control. These cells were negative for production of CD31, CD34,CD45, CD117, CD141, and HLA-DR, DP, DQ, as indicated by fluorescencevalues consistent with the IgG control.

Summary.

Analysis of placenta-derived cells by flow cytometry has established aprofile useful to identify of these cell lines. Placenta-derived cellsare positive for CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-A,B,Cand negative for CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ.This identity was consistent between variations in variables includingthe donor, passage, culture vessel surface coating, digestion enzymes,and placental layer. Some variation in individual fluorescence valuehistogram curve means and ranges were observed, but all positive curvesunder all conditions tested were normal and expressed fluorescencevalues greater than the IgG control, thus confirming that the cellscomprise a homogeneous population which has positive expression of themarkers.

Example 8 Analysis of Placenta-Derived Cells by Affymetrix GeneChip®Arrays

Affymetrix GeneChip® arrays were used to compare gene expressionprofiles of placenta-derived cells with umbilical cord-derived cells,fibroblasts, human mesenchymal stem cells, and another cell line derivedfrom human bone marrow. This analysis provided a characterization of thepostpartum cells and identified unique molecular markers for thesecells.

Materials and Methods

Isolation and Culture of Cells

Postpartum Tissue-Derived Cells.

Human umbilical cords and placenta were obtained from National DiseaseResearch Interchange (NDRI, Philadelphia, Pa.) from normal full termdeliveries with patient consent. The tissues were received and cellswere isolated as described in Example 1. Cells were cultured in GrowthMedium (Dulbecco's Modified Essential Media (DMEM-low glucose;Invitrogen, Carlsbad, Calif.) with 15% (v/v) fetal bovine serum(Hyclone, Logan Utah), 100 Units/milliliter penicillin, 100microgram/milliliter streptomycin (Invitrogen, Carlsbad, Calif.), and0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis Mo.)) on gelatin-coatedtissue culture plastic flasks. The cultures were incubated understandard growth conditions.

Fibroblasts.

Human dermal fibroblasts were purchased from Cambrex Incorporated(Walkersville, Md.; Lot number 9F0844) and were obtained from ATCCCRL-1501 (CCD39SK). Both lines were cultured in DMEM/F12 medium(Invitrogen, Carlsbad, Calif.) with 10% (v/v) fetal bovine serum(Hyclone) and 100 Units/milliliter penicillin, 100 microgram/milliliterstreptomycin (Invitrogen). The cells were grown on standardtissue-treated plastic.

Human Mesenchymal Stem Cells (hMSC).

hMSCs were purchased from Cambrex Incorporated (Walkersville, Md.; Lotnumbers 2F1655, 2F1656 and 2F1657) and cultured according to themanufacturer's specifications in MSCGM Media (Cambrex). The cells weregrown on standard tissue cultured plastic at 37° C. in standardatmosphere with 5% CO₂.

Human Iliac Crest Bone Marrow Cells (ICBM).

Human iliac crest bone marrow was received from NDRI with patientconsent. The marrow was processed according to the method outlined byHo, et al. (International PCT Publication No. WO03/025149). The marrowwas mixed with lysis buffer (155 microMolar NH₄Cl, 10 microMolar KHCO₃,and 0.1 microMolar EDTA, pH 7.2) at a ratio of 1 part bone marrow to 20parts lysis buffer. The cell suspension was vortexed, incubated for 2minutes at ambient temperature, and centrifuged for 10 minutes at 500×g.The supernatant was discarded and the cell pellet was resuspended inMinimal Essential Medium-alpha (Invitrogen) supplemented with 10% (v/v)fetal bovine serum and 4 microMolar glutamine. The cells werecentrifuged, and the cell pellet was resuspended in fresh medium. Theviable mononuclear cells were counted using trypan-blue exclusion(Sigma, St. Louis, Mo.). The mononuclear cells were seeded intissue-cultured plastic flasks at 5×10⁴ cells/cm². The cells wereincubated at 37° C. with 5% CO₂ at either standard atmospheric O₂ or at5% O₂. Cells were cultured for 5 days without a medium change. Media andnon-adherent cells were removed after 5 days of culture. The adherentcells were maintained in culture.

Isolation of mRNA and GeneChip Analysis.

Actively growing cultures of cells were removed from the flasks with acell scraper in cold phosphate buffered saline (PBS). The cells werecentrifuged for 5 minutes at 300×g. The supernatant was removed, and thecells were resuspended in fresh PBS and centrifuged again. Thesupernatant was removed, and the cell pellet was immediately frozen andstored at −80° C. Cellular mRNA was extracted and transcribed into cDNA.cDNA was then transcribed into cRNA and biotin-labeled. Thebiotin-labeled cRNA was hybridized with HG-U133A GENECHIPoligonucleotide array (Affymetrix, Santa Clara Calif.). Thehybridization and data collection was performed according to themanufacturer's specifications. Analyses were performed using“Significance Analysis of Microarrays” (SAM) version 1.21 computersoftware (Stanford University; Tusher, V. G. et al., 2001, Proc. Natl.Acad. Sci. USA 98: 5116-5121).

Results

Fourteen different populations of cells were analyzed in this study. Thecells along with passage information, culture substrate, and culturemedia are listed in Table 8-1.

TABLE 8-1 Cells analyzed by the microarray study. The cell lines arelisted by their identification code along with passage at the time ofanalysis, cell growth substrate, and growth media. Cell PopulationPassage Substrate Media Umbilical (022803) 2 Gelatin DMEM, 15% FBS, BMEUmbilical (042103) 3 Gelatin DMEM, 15% FBS, BME Umbilical (071003) 4Gelatin DMEM, 15% FBS, BME Placenta (042203) 12 Gelatin DMEM, 15% FBS,BME Placenta (042903) 4 Gelatin DMEM, 15% FBS, BME Placenta (071003) 3Gelatin DMEM, 15% FBS, BME ICBM (070203) (5% O₂) 3 Plastic MEM 10% FBSICBM (062703) (std O₂) 5 Plastic MEM 10% FBS ICBM (062703)(5% O₂) 5Plastic MEM 10% FBS hMSC (Lot 2F1655) 3 Plastic MSCGM hMSC (Lot 2F1656)3 Plastic MSCGM hMSC (Lot 2F1657) 3 Plastic MSCGM hFibroblast (9F0844) 9Plastic DMEM-F12, 10% FBS hFibroblast (ATCC 4 Plastic DMEM-F12, 10% FBSCRL-1501)

The data were evaluated by a Principle Component Analysis, analyzing the290 genes that were differentially expressed in the cells. This analysisallows for a relative comparison for the similarities between thepopulations. Table 8-2 shows the Euclidean distances that werecalculated for the comparison of the cell pairs. The Euclidean distanceswere based on the comparison of the cells based on the 290 genes thatwere differentially expressed among the cell types. The Euclideandistance is inversely proportional to similarity between the expressionof the 290 genes.

TABLE 8-2 The Euclidean Distances for the Cell Pairs. The Euclideandistance was calculated for the cell types using the 290 genes that weredifferentially expressed between the cell types. Similarity between thecells is inversely proportional to the Euclidean distance. Cell PairEuclidean Distance ICBM-hMSC 24.71 Placenta-umbilical 25.52ICBM-Fibroblast 36.44 ICBM-placenta 37.09 Fibroblast-MSC 39.63ICBM-Umbilical 40.15 Fibroblast-Umbilical 41.59 MSC-Placenta 42.84MSC-Umbilical 46.86 ICBM-placenta 48.41

Tables 8-3, 8-4, and 8-5 show the expression of genes increased inplacenta-derived cells (Table 8-3), increased in umbilical cord-derivedcells (Table 8-4), and reduced in umbilical cord- and placenta-derivedcells (Table 8-5). The column entitled “Probe Set ID” refers to themanufacturer's identification code for the sets of severaloligonucleotide probes located on a particular site on the chip, whichhybridize to the named gene (column “Gene Name”), comprising a sequencethat can be found within the NCBI (GenBank) database at the specifiedaccession number (column “NCBI Accession Number”).

TABLE 8-3 Genes shown to have specifically increased expression in theplacenta-derived cells as compared to the other cell lines assayed.Genes Increased in Placenta-Derived Cells NCBI Accession Probe Set IDGene Name Number 209732_at C-type (calcium dependent, AF070642carbohydrate-recognition domain) lectin, superfamily member 2(activation-induced) 206067_s_at Wilms tumor 1 NM_024426 207016_s_ataldehyde dehydrogenase 1 family, AB015228 member A2 206367_at reninNM_000537 210004_at oxidised low density lipoprotein AF035776(lectin-like) receptor 1 214993_at Homo sapiens, clone IMAGE: AF0706424179671, mRNA, partial cds 202178_at protein kinase C, zeta NM_002744209780_at hypothetical protein DKFZp564F013 AL136883 204135_atdownregulated in ovarian cancer 1 NM_014890 213542_at Homo sapiens mRNA;cDNA AI246730 DKFZp547K1113 (from clone DKFZp547K1113)

TABLE 8-4 Genes shown to have specifically increased expression inumbilical cord-derived cells as compared to the other cell linesassayed. Genes Increased in Umbilical Cord-Derived Cells NCBI AccessionProbe Set ID Gene Name Number 202859_x_at interleukin 8 NM_000584211506_s_at interleukin 8 AF043337 210222_s_at reticulon 1 BC000314204470_at chemokine (C-X-C motif) ligand 1 NM_001511 (melanoma growthstimulating activity 206336_at chemokine (C-X-C motif) ligand 6NM_002993 (granulocyte chemotactic protein 2) 207850_at chemokine (C-X-Cmotif) ligand 3 NM_002090 203485_at reticulon 1 NM_021136 202644_s_attumor necrosis factor, alpha-induced NM_006290 protein 3

TABLE 8-5 Genes that were shown to have decreased expression in theumbilical cord and placenta cells as compared to the other cell linesassayed. Genes Decreased in Umbilical Cord- and Placenta-Derived CellsNCBI Accession Probe Set ID Gene name Number 210135_s_at short staturehomeobox 2 AF022654.1 205824_at heat shock 27 kDa protein 2 NM_001541.1209687_at chemokine (C-X-C motif) ligand 12 (stromal cell- U19495.1derived factor 1) 203666_at chemokine (C-X-C motif) ligand 12 (stromalcell- NM_000609.1 derived factor 1) 212670_at elastin (supravalvularaortic stenosis, Williams- AA479278 Beuren syndrome) 213381_at Homosapiens mRNA; cDNA DKFZp586M2022 N91149 (from clone DKFZp586M2022)206201_s_at mesenchyme homeobox 2 (growth arrest-specific NM_005924.1homeobox) 205817_at sine oculis homeobox homolog 1 (Drosophila)NM_005982.1 209283_at crystallin, alpha B AF007162.1 212793_atdishevelled associated activator of morphogenesis BF513244 2 213488_atDKFZP586B2420 protein AL050143.1 209763_at similar to neuralin 1AL049176 205200_at tetranectin (plasminogen binding protein) NM_003278.1205743_at src homology three (SH3) and cysteine rich NM_003149.1 domain200921_s_at B-cell translocation gene 1, anti-proliferative NM_001731.1206932_at cholesterol 25-hydroxylase NM_003956.1 204198_s_atrunt-related transcription factor 3 AA541630 219747_at hypotheticalprotein FLJ23191 NM_024574.1 204773_at interleukin 11 receptor, alphaNM_004512.1 202465_at procollagen C-endopeptidase enhancer NM_002593.2203706_s_at frizzled homolog 7 (Drosophila) NM_003507.1 212736_athypothetical gene BC008967 BE299456 214587_at collagen, type VIII, alpha1 BE877796 201645_at tenascin C (hexabrachion) NM_002160.1 210239_atiroquois homeobox protein 5 U90304.1 203903_s_at hephaestin NM_014799.1205816_at integrin, beta 8 NM_002214.1 203069_at synaptic vesicleglycoprotein 2 NM_014849.1 213909_at Homo sapiens cDNA FLJ12280 fis,clone AU147799 MAMMA1001744 206315_at cytokine receptor-like factor 1NM_004750.1 204401_at potassium intermediate/small conductanceNM_002250.1 calcium-activated channel, subfamily N, member 4 216331_atintegrin, alpha 7 AK022548.1 209663_s_at integrin, alpha 7 AF072132.1213125_at DKFZP586L151 protein AW007573 202133_at transcriptionalco-activator with PDZ-binding AA081084 motif (TAZ) 206511_s_at sineoculis homeobox homolog 2 (Drosophila) NM_016932.1 213435_at KIAA1034protein AB028957.1 206115_at early growth response 3 NM_004430.1213707_s_at distal-less homeobox 5 NM_005221.3 218181_s_at hypotheticalprotein FLJ20373 NM_017792.1 209160_at aldo-keto reductase family 1,member C3 (3-alpha AB018580.1 hydroxysteroid dehydrogenase, type II)213905_x_at biglycan AA845258 201261_x_at biglycan BC002416.1 202132_attranscriptional co-activator with PDZ-binding AA081084 motif (TAZ)214701_s_at fibronectin 1 AJ276395.1 213791_at proenkephalin NM_006211.1205422_s_at integrin, beta-like 1 (with EGF-like repeat NM_004791.1domains) 214927_at Homo sapiens mRNA full length insert cDNA AL359052.1clone EUROIMAGE 1968422 206070_s_at EphA3 AF213459.1 212805_at KIAA0367protein AB002365.1 219789_at natriuretic peptide receptor C/guanylatecyclase C AI628360 (atrionatriuretic peptide receptor C) 219054_athypothetical protein FLJ14054 NM_024563.1 213429_at Homo sapiens mRNA;cDNA DKFZp564B222 AW025579 (from clone DKFZp564B222) 204929_s_atvesicle-associated membrane protein 5 NM_006634.1 (myobrevin)201843_s_at EGF-containing fibulin-like extracellular matrix NM_004105.2protein 1 221478_at BCL2/adenovirus E1B 19 kDa interacting proteinAL132665.1 3-like 201792_at AE binding protein 1 NM_001129.2 204570_atcytochrome c oxidase subunit VIIa polypeptide 1 NM_001864.1 (muscle)201621_at neuroblastoma, suppression of tumorigenicity 1 NM_005380.1202718_at insulin-like growth factor binding protein 2, NM_000597.1 36kDa

Tables 8-6, 8-7, and 8-8 show the expression of genes increased in humanfibroblasts (Table 8-6), ICBM cells (Table 8-7), and MSCs (Table 8-8).

TABLE 8-6 Genes that were shown to have increased expression infibroblasts as compared to the other cell lines assayed. Genes increasedin fibroblasts dual specificity phosphatase 2 KIAA0527 protein Homosapiens cDNA: FLJ23224 fis, clone ADSU02206 dynein, cytoplasmic,intermediate polypeptide 1 ankyrin 3, node of Ranvier (ankyrin G)inhibin, beta A (activin A, activin AB alpha polypeptide) ectonucleotidepyrophosphatase/phosphodiesterase 4 (putative function) KIAA1053 proteinmicrotubule-associated protein 1A zinc finger protein 41 HSPC019 proteinHomo sapiens cDNA: FLJ23564 fis, clone LNG10773 Homo sapiens mRNA; cDNADKFZp564A072 (from clone DKFZp564A072) LIM protein (similar to ratprotein kinase C-binding enigma) inhibitor of kappa light polypeptidegene enhancer in B-cells, kinase complex-associated protein hypotheticalprotein FLJ22004 Human (clone CTG-A4) mRNA sequence ESTs, Moderatelysimilar to cytokine receptor-like factor 2; cytokine receptor CRL2precursor [Homo sapiens] transforming growth factor, beta 2 hypotheticalprotein MGC29643 antigen identified by monoclonal antibody MRC OX-2

TABLE 8-7 Genes that were shown to have increased expression in theICBM-derived cells as compared to the other cell lines assayed. GenesIncreased In ICBM Cells cardiac ankyrin repeat protein MHC class Iregion ORF integrin, alpha 10 hypothetical protein FLJ22362UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3) interferon-induced protein44 SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomalsex-reversal) keratin associated protein 1-1 hippocalcin-like 1 jagged 1(Alagille syndrome) proteoglycan 1, secretory granule

TABLE 8-8 Genes that were shown to have increased expression in the MSCcells as compared to the other cell lines assayed. Genes Increased InMSC Cells interleukin 26 maltase-glucoamylase (alpha-glucosidase)nuclear receptor subfamily 4, group A, member 2 v-fos FBJ murineosteosarcoma viral oncogene homolog hypothetical protein DC42 nuclearreceptor subfamily 4, group A, member 2 FBJ murine osteosarcoma viraloncogene homolog B WNT1 inducible signaling pathway protein 1 MCF.2 cellline derived transforming sequence potassium channel, subfamily K,member 15 cartilage paired-class homeoprotein 1 Homo sapiens cDNAFLJ12232 fis, clone MAMMA1001206 Homo sapiens cDNA FLJ34668 fis, cloneLIVER2000775 jun B proto-oncogene B-cell CLL/lymphoma 6 (zinc fingerprotein 51) zinc finger protein 36, C3H type, homolog (mouse)

Summary.

The GENECHIP analysis was performed to provide a molecularcharacterization of the postpartum cells derived from placenta. Thisanalysis included cells derived from three different placentas. Thestudy also included three different lines of umbilical cord-derivedcells, two different lines of dermal fibroblasts, three lines ofmesenchymal stem cells, and three lines of iliac crest bone marrowcells. The mRNA that was expressed by these cells was analyzed byAffyMetrix GENECHIP that contained oligonucleotide probes for 22,000genes.

Results showed that 290 genes are differentially expressed in these fivedifferent cell types. These genes include ten genes that arespecifically increased in the placenta-derived cells. Fifty-four geneswere found to have specifically lower expression levels in placenta.

The expression of selected genes has been confirmed by PCR in Example 9.These results demonstrate that the placenta-derived cells have adistinct gene expression profile, for example, as compared to bonemarrow-derived cells and fibroblasts.

Example 9 Cell Markers in Placenta-Derived Cells

Similarities and differences in gene expression between cells derivedfrom the human placenta and cells derived from other sources wereassessed by comparing their gene expression profiles using an AffymetrixGenechip. Six “signature” genes were identified: oxidized LDL receptor1, interleukin-8, renin, reticulon, chemokine receptor ligand 3 (CXCligand 3), and granulocyte chemotactic protein 2 (GCP-2). These“signature” genes were expressed at relatively high levels inplacenta-derived cells. This analysis was conducted to verify themicroarray data and find accordance/divergence between gene and proteinexpression, as well as to establish a series of reliable assay fordetection of unique identifiers for placenta-derived cells.

Methods & Materials

Cells.

Placenta-derived cells (three isolates, including one isolatepredominately neonatal as identified by karyotyping analysis) and NormalHuman Dermal Fibroblasts (NHDF; neonatal and adult) were grown in Growthmedium (DMEM-low glucose (Gibco, Carlsbad, Calif.), 15% (v/v) fetalbovine serum (Cat. #SH30070.03; Hyclone, Logan, Utah), 0.001% (v/v)beta-mercaptoethanol (Sigma, St. Louis, Mo.), 50 Units/milliliterpenicillin, 50 microgram/milliliter streptomycin (Gibco, Carlsbad,Calif.)) in a gelatin-coated T75 flask. Mesenchymal Stem Cells (MSCs)were grown in a Mesenchymal Stem Cell Growth Medium Bullet kit (MSCGM;Cambrex, Walkerville, Md.).

For IL-8 experiments, cells were thawed from liquid nitrogen and platedin gelatin-coated flasks at 5,000 cells/cm², grown for 48 hours inGrowth medium and then grown for 8 hours in 10 milliliters of serumstarvation medium [DMEM-low glucose (Gibco, Carlsbad, Calif.), 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin(Gibco, Carlsbad, Calif.) and 0.1% (w/v) Bovine Serum Albumin (BSA;Sigma, St. Louis, Mo.)]. After this treatment, RNA was extracted and thesupernatants were centrifuged at 150×g for 5 minutes to remove cellulardebris.

Cell Culture for ELISA Assay.

Placenta-derived cells and human fibroblasts derived from human neonatalforeskin were cultured in Growth medium in gelatin-coated T75 flasks.Cells were frozen at passage 11 in liquid nitrogen. Cells were thawedand transferred to 15 milliliter centrifuge tubes. After centrifugationat 150×g for 5 minutes, the supernatant was discarded. Cells wereresuspended in 4 milliliters culture medium and counted. Cells weregrown in a 75 cm² flask containing 15 milliliters of Growth medium at375,000 cell/flask for 24 hours. The medium was changed to a serumstarvation medium for 8 hours. Serum starvation medium was collected atthe end of incubation, centrifuged at 14,000×g for 5 minutes, and storedat −20° C.

To estimate the number of cells in each flask, 2 milliliters oftrypsin/EDTA (Gibco, Carlsbad, Calif.) was added to each flask. Aftercells detached from the flask, trypsin activity was neutralized with 8milliliters of Growth medium. Cells were transferred to a 15 millilitercentrifuge tube and centrifuged at 150×g for 5 minutes. Supernatant wasremoved and 1 milliliter Growth medium was added to each tube toresuspend the cells. Cell number was estimated using a hemocytometer.

ELISA Assay.

The amount of IL-8 secreted by the cells into serum starvation mediumwas analyzed using ELISA assays (R&D Systems, Minneapolis, Minn.). Allassays were tested according to the instructions provided by themanufacturer.

Total RNA Isolation.

RNA was extracted from confluent placenta-derived cells and fibroblastsor for IL-8 expression from cells treated as described above. Cells werelysed with 350 microliter buffer RLT containing beta-mercaptoethanol(Sigma, St. Louis, Mo.) according to the manufacturer's instructions(RNeasy Mini Kit; Qiagen, Valencia, Calif.). RNA was extracted accordingto the manufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia,Calif.) and subjected to DNase treatment (2.7 U/sample) (Sigma St.Louis, Mo.). RNA was eluted with 50 microliter DEPC-treated water andstored at −80° C. RNA was extracted from human placenta. Tissue (30milligram) was suspended in 700 microliter of buffer RLT containingbeta-mercaptoethanol. Samples were mechanically homogenized, and the RNAextraction proceeded according to manufacturer's specification. RNA wasextracted with 50 microliter of DEPC-treated water and stored at −80° C.

Reverse Transcription.

RNA was reverse transcribed using random hexamers with the TaqManreverse transcription reagents (Applied Biosystems, Foster City, Calif.)at 25° C. for 10 minutes, 37° C. for 60 minutes, and 95° C. for 10minutes. Samples were stored at −20° C.

Genes identified by cDNA microarray as uniquely regulated in postpartumcells (“signature genes,” including oxidized LDL receptor,interleukin-8, renin, and reticulon), were further investigated usingreal-time and conventional PCR.

Real-Time PCR.

PCR was performed on cDNA samples using ASSAYS-ON-DEMAND gene expressionproducts: oxidized LDL receptor (Hs00234028); renin (Hs00166915);reticulon (Hs00382515); CXC ligand 3 (Hs00171061); GCP-2 (Hs00605742);IL-8 (Hs00174103); and GAPDH were mixed with cDNA and TaqMan UniversalPCR master mix according to the manufacturer's instructions (AppliedBiosystems, Foster City, Calif.) using a 7000 sequence detection systemwith ABI Prism 7000 SDS software (Applied Biosystems, Foster City,Calif.). Thermal cycle conditions were initially 50° C. for 2 minute and95° C. for 10 minute, followed by 40 cycles of 95° C. for 15 second and60° C. for 1 minute. PCR data was analyzed according to manufacturer'sspecifications (User Bulletin #2 from Applied Biosystems for ABI Prism7700 Sequence Detection System).

Conventional PCR.

Conventional PCR was performed using an ABI PRISM 7700 (Perkin ElmerApplied Biosystems, Boston, Mass.) to confirm the results from real-timePCR. PCR was performed using 2 microliter of cDNA solution, 1×TAQpolymerase (tradename AMPLITAQ GOLD) universal mix PCR reaction buffer(Applied Biosystems, Foster City, Calif.) and initial denaturation at94° C. for 5 minutes. Amplification was optimized for each primer set.For IL-8, CXC ligand 3, and reticulon (94° C. for 15 seconds, 55° C. for15 seconds, and 72° C. for 30 seconds for 30 cycles); for renin (94° C.for 15 seconds, 53° C. for 15 seconds, and 72° C. for 30 seconds for 38cycles); for oxidized LDL receptor and GAPDH (94° C. for 15 seconds, 55°C. for 15 seconds, and 72° C. for 30 seconds for 33 cycles). Primersused for amplification are listed in Table 9-1. Primer concentration inthe final PCR reaction was 1 microMolar except for GAPDH which was 0.5microMolar. GAPDH primers were the same as real-time PCR, except thatthe manufacturer's TaqMan probe was not added to the final PCR reaction.Samples were run on 2% (w/v) agarose gel and stained with ethidiumbromide (Sigma, St. Louis, Mo.). Images were captured using a 667Universal Twinpack film (VWR International, South Plainfield, N.J.)using a focal-length POLAROID camera (VWR International, SouthPlainfield, N.J.).

TABLE 9-1 Primers used Primer name Primers Oxidized S: 5′-GAGAAATCCAAAGAGCAAATGG-3′ LDL receptor (SEQ ID NO: 1)A: 5′-AGAATGGAAAACTGGAATAGG-3′ (SEQ ID NO: 2) ReninS: 5′-TCTTCGATGCTTCGGATTCC-3′ (SEQ ID NO: 3)A: 5′-GAATTCTCGGAATCTCTGTTG-3′ (SEQ ID NO: 4) ReticulonS: 5′-TTACAAGCAGTGCAGAAAACC-3′ (SEQ ID NO: 5)A: 5′-AGTAAACATTGAAACCACAGCC-3′ (SEQ ID NO: 6) Interleukin-8S: 5′-TCTGCAGCTCTGTGTGAAGG-3′ (SEQ ID NO: 7)A: 5′-CTTCAAAAACTTCTCCACAACC-3′ (SEQ ID NO: 8) ChemokineS: 5′-CCCACGCCACGCTCTCC-3′ (CXC) ligand 3 (SEQ ID NO: 9)A: 5′-TCCTGTCAGTTGGTGCTCC-3′ (SEQ ID NO: 10)

Immunofluorescence.

Cells were fixed with cold 4% (w/v) paraformaldehyde (Sigma-Aldrich, St.Louis, Mo.) for 10 minutes at room temperature. Placenta-derived cellsat passage 0 (P0) (one isolate, directly after isolation) and passage 11(P11) (two isolates) and fibroblasts (P11) were used.Immunocytochemistry was performed using antibodies directed against thefollowing epitopes: vimentin (1:500, Sigma, St. Louis, Mo.), desmin(1:150; Sigma—raised against rabbit; or 1:300; Chemicon, Temecula,Calif.—raised against mouse,), alpha-smooth muscle actin (SMA; 1:400;Sigma), cytokeratin 18 (CK18; 1:400; Sigma), von Willebrand Factor (vWF;1:200; Sigma), and CD34 (human CD34 Class III; 1:100; DAKOCytomation,Carpinteria, Calif.). In addition, the following markers were tested onpassage 11 placenta-derived cells: anti-human GROalpha-PE (1:100; BectonDickinson, Franklin Lakes, N.J.), anti-human GCP-2 (1:100; Santa CruzBiotech, Santa Cruz, Calif.), anti-human oxidized LDL receptor 1 (ox-LDLR1; 1:100; Santa Cruz Biotech), and anti-human NOGA-A (1:100; SantaCruz, Biotech).

Cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma, St. Louis, Mo.) for 30 minutes to access intracellular antigens.Where the epitope of interest was located on the cell surface (CD34,ox-LDL R1), Triton X-100 was omitted in all steps of the procedure inorder to prevent epitope loss. Furthermore, in instances where theprimary antibody was raised against goat (GCP-2, ox-LDL R1, NOGO-A), 3%(v/v) donkey serum was used in place of goat serum throughout theprocess. Primary antibodies, diluted in blocking solution, were thenapplied to the cultures for a period of 1 hour at room temperature. Theprimary antibody solutions were removed, and the cultures were washedwith PBS prior to application of secondary antibody solutions (1 hour atroom temperature) containing block along with goat anti-mouse IgG—TexasRed (1:250; Molecular Probes, Eugene, Oreg.) and/or goat anti-rabbitIgG—Alexa 488 (1:250; Molecular Probes) or donkey anti-goat IgG—FITC(1:150, Santa Cruz Biotech). Cultures were washed and 10 microMolar DAPI(Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using anappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed, with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Preparation of Cells for FACS Analysis.

Adherent cells in flasks were washed in phosphate buffered saline (PBS)(Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco,Carlsbad, Calif.). Cells were harvested, centrifuged, and re-suspendedin 3% (v/v) FBS in PBS at a cell concentration of 1×10⁷/milliliter. Onehundred microliter aliquots were delivered to conical tubes. Cellsstained for intracellular antigens were permeabilized with Perm/Washbuffer (BD Pharmingen, San Diego, Calif.). Antibody was added toaliquots as per manufacturer's specifications and the cells wereincubated in the dark for 30 minutes at 4° C. After incubation, cellswere washed with PBS and centrifuged to remove excess antibody. Cellsrequiring a secondary antibody were resuspended in 100 microliter of 3%FBS. Secondary antibody was added as per manufacturer's specificationand the cells were incubated in the dark for 30 minutes at 4° C. Afterincubation, cells were washed with PBS and centrifuged to remove excesssecondary antibody. Washed cells were resuspended in 0.5 milliliter PBSand analyzed by flow cytometry. The following antibodies were used:oxidized LDL receptor 1 (sc-5813; Santa Cruz, Biotech), GROa (555042; BDPharmingen, Bedford, Mass.), Mouse IgG1 kappa, (P-4685 and M-5284;Sigma), Donkey against Goat IgG (sc-3743; Santa Cruz, Biotech.).

FACS Analysis.

Flow cytometry analysis was performed with FACScalibur (Becton DickinsonSan Jose, Calif.).

Results

Results of real-time PCR for selected “signature” genes performed oncDNA from cells derived from human placentas, adult and neonatalfibroblasts, and Mesenchymal Stem Cells (MSCs) indicate that bothoxidized LDL receptor and renin were expressed at higher level in theplacenta-derived cells as compared to other cells. The data obtainedfrom real-time PCR were analyzed by the ΔΔC_(T) method and expressed ona logarithmic scale. No significant difference in the expression levelsof CXC ligand 3 and GCP-2 were found between placenta-derived cells andcontrols. CXC ligand 3 was expressed at very low levels. GCP-2 wasexpressed at levels comparable to human adult and neonatal fibroblasts.The results of real-time PCR were confirmed by conventional PCR.Sequencing of PCR products further validated these observations. Nosignificant difference in the expression level of CXC ligand 3 was foundbetween postpartum cells and controls using conventional PCR CXC ligand3 primers listed in Table 9-1.

The production of the cytokine IL-8 in placenta-derived cells iselevated in both Growth medium-cultured and serum-starvedplacenta-derived cells. All real-time PCR data was validated withconventional PCR and by sequencing PCR products.

When supernatants of cells grown in serum-free medium were examined forthe presence of IL-8, high amounts were detected in media derived fromcertain isolates of placenta cells (Table 9-2). No IL-8 was detected inmedium derived from human dermal fibroblasts.

TABLE 9-2 IL-8 protein production measured by ELISA Cell type IL-8 HumanFibroblasts ND Placenta Isolate 1 ND Placenta Isolate 2 ND PlacentaIsolate3 (normal O₂)  17.27 ± 8.63 Placenta Isolate 3 (lowO₂, W/O BME)264.92 ± 9.88 Results of the ELISA assay for interleukin-8 (IL-8)performed on placenta-derived cells and human skin fibroblasts. Valuesare presented here are picogram/million cells, n = 2, sem. ND: NotDetected

Placenta-derived cells were examined for the production of oxidized LDLreceptor, GCP-2, and GROalpha by FACS analysis. Cells tested positivefor GCP-2. Oxidized LDL receptor and GROalpha were not detected by thismethod.

Placenta-derived cells were tested for the production of selectedproteins by immunocytochemical analysis. Immediately after isolation(passage 0), cells derived from the human placenta were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Cells stained positive for both alpha-smooth muscleactin and vimentin. This pattern was preserved through passage 11. Onlya few cells (<5%) at passage 0 stained positive for cytokeratin 18.

Placenta-derived cells at passage 11 were also investigated byimmunocytochemistry for the production of GROalpha and GCP-2.Placenta-derived cells were GCP-2 positive, but GROalpha production wasnot detected by this method.

Summary.

Accordance between gene expression levels measured by microarray and PCR(both real-time and conventional) has been established for four genes:oxidized LDL receptor 1, renin, reticulon, and IL-8. The expression ofthese genes was differentially regulated at the mRNA level inplacenta-derived cells, with IL-8 also differentially regulated at theprotein level. The presence of oxidized LDL receptor was not detected atthe protein level by FACS analysis in cells derived from the placenta.Differential expression of GCP-2 and CXC ligand 3 was not confirmed atthe mRNA level, however, GCP-2 was detected at the protein level by FACSanalysis in the placenta-derived cells. Although this result may not befully consistent with data obtained from the microarray experiment, anyinconsistency may simply be due to differences in the sensitivity of themethodologies.

Immediately after isolation (passage 0), cells derived from the humanplacenta stained positive for both alpha-smooth muscle actin andvimentin. This pattern was also observed in cells at passage 11. Theseresults suggest that vimentin and alpha-smooth muscle actin productionis preserved in cells with passaging, for example, in the Growth mediumused here.

Example 10 Immunohistochemical Characterization of PDC Phenotype

The phenotypes of cells found within human placental tissue was analyzedby immunohistochemistry.

Materials & Methods

Tissue Preparation.

Human placenta tissue was harvested and immersion-fixed in 4% (w/v)paraformaldehyde overnight at 4° C. Immunohistochemistry was performedusing antibodies directed against the following epitopes (see Table10-1): vimentin (1:500; Sigma, St. Louis, Mo.), desmin (1:150, raisedagainst rabbit; Sigma; or 1:300, raised against mouse; Chemicon,Temecula, Calif.), alpha-smooth muscle actin (SMA; 1:400; Sigma),cytokeratin 18 (CK18; 1:400; Sigma), von Willebrand Factor (vWF; 1:200;Sigma), and CD34 (human CD34 Class III; 1:100; DAKOCytomation,Carpinteria, Calif.). In addition, the following markers were tested:anti-human GROalpha-PE (1:100; Becton Dickinson, Franklin Lakes, N.J.),anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, Calif.),anti-human oxidized LDL receptor 1 (ox-LDL R1; 1:100; Santa CruzBiotech), and anti-human NOGO-A (1:100; Santa Cruz Biotech). Fixedspecimens were trimmed with a scalpel and placed within OCT embeddingcompound (Tissue-Tek OCT; Sakura, Torrance, Calif.) on a dry ice bathcontaining ethanol. Frozen blocks were then sectioned (10 micron thick)using a standard cryostat (Leica Microsystems) and mounted onto glassslides for staining.

TABLE 10-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Vimentin 1:500 Sigma, St. Louis, MO Desmin (rb) 1:150 SigmaDesmin (m) 1:300 Chemicon, Temecula, CA alpha-smooth muscle actin 1:400Sigma (SMA) Cytokeratin 18 (CK18) 1:400 Sigma von Willebrand factor1:200 Sigma (vWF) CD34 III 1:100 DakoCytomation, Carpinteria, CAGROalpha - PE 1:100 BD, Franklin Lakes, NJ GCP-2 1:100 Santa CruzBiotech Ox-LDL R1 1:100 Santa Cruz Biotech NOGO-A 1:100 Santa CruzBiotech

Immunohistochemistry.

Immunohistochemistry was performed similar to previous studies (e.g.,Messina, et al. (2003) Exper. Neurol. 184: 816-829). Tissue sectionswere washed with phosphate-buffered saline (PBS) and exposed to aprotein blocking solution containing PBS, 4% (v/v) goat serum (Chemicon,Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 1hour to access intracellular antigens. In instances where the epitope ofinterest would be located on the cell surface (CD34, ox-LDL R1), tritonwas omitted in all steps of the procedure in order to prevent epitopeloss. Furthermore, in instances where the primary antibody was raisedagainst goat (GCP-2, ox-LDL R1, NOGO-A), 3% (v/v) donkey serum was usedin place of goat serum throughout the procedure. Primary antibodies,diluted in blocking solution, were then applied to the sections for aperiod of 4 hours at room temperature. Primary antibody solutions wereremoved, and cultures washed with PBS prior to application of secondaryantibody solutions (1 hour at room temperature) containing block alongwith goat anti-mouse IgG—Texas Red (1:250; Molecular Probes, Eugene,Oreg.) and/or goat anti-rabbit IgG—Alexa 488 (1:250; Molecular Probes)or donkey anti-goat IgG—FITC (1:150; Santa Cruz Biotech). Cultures werewashed, and 10 microMolar DAPI (Molecular Probes) was applied for 10minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). Positive staining was representedby fluorescence signal above control staining. Representative imageswere captured using a digital color videocamera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

Results

Placenta Characterization.

Vimentin, desmin, SMA, CK18, vWF, and CD34 were all observed within theplacenta and regionally specific.

GROalpha, GCP-2, ox-LDL R1, and NOGO-A Tissue Expression.

None of these markers were observed within placental tissue.

Summary.

Vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18, vonWillebrand Factor, and CD34 are expressed in cells within humanplacenta.

Example 11 In Vitro Immunology

Postpartum cell lines were evaluated in vitro for their immunologicalcharacteristics in an effort to predict the immunological response, ifany, these cells would elicit upon in vivo transplantation. Postpartumcell lines were assayed by flow cytometry for the production of HLA-DR,HLA-DP, HLA-DQ, CD80, CD86, and B7-H2. These proteins are expressed byantigen-presenting cells (APC) and are required for the directstimulation of naïve CD4⁺ T cells (Abbas & Lichtman, CELLULAR ANDMOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171).The cell lines were also analyzed by flow cytometry for the productionof HLA-G (Abbas & Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th Ed.(2003) Saunders, Philadelphia, p. 171), CD 178 (Coumans, et. al., (1999)Journal of Immunological Methods 224, 185-196), and PD-L2 (Abbas &Lichtman, CELLULAR AND MOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders,Philadelphia, p. 171; Brown, et. al. (2003) The Journal of Immunology170, 1257-1266). The production of these proteins by cells residing inplacental tissues is thought to mediate the immuno-privileged status ofplacental tissues in utero. To predict the extent to whichplacenta-derived cell lines elicit an immune response in vivo, the celllines were tested in a one-way mixed lymphocyte reaction (MLR).

Materials and Methods

Cell Culture.

Cells were cultured in Growth Medium (DMEM-low glucose (Gibco, Carlsbad,Calif.), 15% (v/v) fetal bovine serum (FBS); (Hyclone, Logan, Utah),0.001% (v/v) betamercaptoethanol (Sigma, St. Louis, Mo.), 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin(Gibco, Carlsbad, Calif.)) until confluent in T75 flasks (Corning,Corning, N.Y.) coated with 2% gelatin (Sigma, St. Louis, Mo.).

Antibody Staining.

Cells were washed in phosphate buffered saline (PBS) (Gibco, Carlsbad,Calif.) and detached with Trypsin/EDTA (Gibco, Carlsbad, Calif.). Cellswere harvested, centrifuged, and re-suspended in 3% (v/v) FBS in PBS ata cell concentration of 1×10⁷ per milliliter. Antibody (Table 11-1) wasadded to one hundred microliters of cell suspension as permanufacturer's specifications and incubated in the dark for 30 minutesat 4° C. After incubation, cells were washed with PBS and centrifuged toremove unbound antibody. Cells were re-suspended in five hundredmicroliters of PBS and analyzed by flow cytometry using a FACSCaliburinstrument (Becton Dickinson, San Jose, Calif.).

TABLE 11-1 Antibodies Catalog Antibody Manufacturer Number HLA-DRDPDQ BDPharmingen (San Diego, CA) 555558 CD80 BD Pharmingen (San Diego, CA)557227 CD86 BD Pharmingen (San Diego, CA) 555665 B7-H2 BD Pharmingen(San Diego, CA) 552502 HLA-G Abcam (Cambridgeshire, UK) ab 7904-100 CD178 Santa Cruz (San Cruz, CA) sc-19681 PD-L2 BD Pharmingen (San Diego,CA) 557846 Mouse IgG2a Sigma (St. Louis, MO) F-6522 Mouse IgG1kappaSigma (St. Louis, MO) P-4685

Mixed Lymphocyte Reaction.

Cryopreserved vials of passage 11 placenta-derived PDCs labeled as cellline B were sent on dry ice to CTBR (Senneville, Quebec) to conduct amixed lymphocyte reaction using CTBR SOP no. CAC-031. Peripheral bloodmononuclear cells (PBMCs) were collected from multiple male and femalevolunteer donors. Stimulator (donor) allogeneic PBMC, autologous PBMC,and placenta-derived cell lines were treated with mitomycin C.Autologous and mitomycin C-treated stimulator cells were added toresponder (recipient) PBMCs and cultured for 4 days. After incubation,[³H]thymidine was added to each sample and cultured for 18 hours.Following harvest of the cells, radiolabeled DNA was extracted, and[³H]-thymidine incorporation was measured using a scintillation counter.

The stimulation index for the allogeneic donor (SIAD) was calculated asthe mean proliferation of the receiver plus mitomycin C-treatedallogeneic donor divided by the baseline proliferation of the receiver.The stimulation index of the placenta-derived cell was calculated as themean proliferation of the receiver plus mitomycin C-treatedplacenta-derived cell line divided by the baseline proliferation of thereceiver.

Results

Mixed Lymphocyte Reaction-Placenta.

Seven human volunteer blood donors were screened to identify a singleallogeneic donor that would exhibit a robust proliferation response in amixed lymphocyte reaction with the other six blood donors. This donorwas selected as the allogeneic positive control donor. The remaining sixblood donors were selected as recipients. The allogeneic positivecontrol donor and placenta cell lines were treated with mitomycin C andcultured in a mixed lymphocyte reaction with the six individualallogeneic receivers. Reactions were performed in triplicate using twocell culture plates with three receivers per plate (Table 11-2). Theaverage stimulation index ranged from 1.3 (plate 2) to 3 (plate 1) andthe allogeneic donor positive controls ranged from 46.25 (plate 2) to279 (plate 1) (Table 11-3).

TABLE 11-2 Mixed Lymphocyte Reaction Data - Cell Line B (Placenta) DPMfor Proliferation Assay Replicates Analytical number Culture System 1 23 Mean SD CV Plate ID: Plate1 IM03-7769 Proliferation baseline ofreceiver 79 119 138 112.0 30.12 26.9 Control of autostimulation(Mitomycin C treated autologous cells) 241 272 175 229.3 49.54 21.6 MLRallogenic donor IM03-7768 (Mitomycin C treated) 23971 22352 2092122414.7 1525.97 6.8 MLR with cell line (Mitomycin C treated cell type B)664 559 1090 771.0 281.21 36.5 SI (donor) 200 SI (cell line) 7 IM03-7770Proliferation baseline of receiver 206 134 262 200.7 64.17 32.0 Controlof autostimulation (Mitomycin C treated autologous cells) 1091 602 524739.0 307.33 41.6 MLR allogenic donor IM03-7768 (Mitomycin C treated)45005 43729 44071 44268.3 660.49 1.5 MLR with cell line (Mitomycin Ctreated cell type B) 533 2582 2376 1830.3 1128.24 61.6 SI (donor) 221 SI(cell line) 9 IM03-7771 Proliferation baseline of receiver 157 87 128124.0 35.17 28.4 Control of autostimulation (Mitomycin C treatedautologous cells) 293 138 508 313.0 185.81 59.4 MLR allogenic donorIM03-7768 (Mitomycin C treated) 24497 34348 31388 30077.7 5054.53 16.8MLR with cell line (Mitomycin C treated cell type B) 601 643 a 622.029.70 4.8 SI (donor) 243 SI (cell line) 5 IM03-7772 Proliferationbaseline of receiver 56 98 51 68.3 25.81 37.8 Control of autostimulation(Mitomycin C treated autologous cells) 133 120 213 155.3 50.36 32.4 MLRallogenic donor IM03-7768 (Mitomycin C treated) 14222 20076 2216818822.0 4118.75 21.9 MLR with cell line (Mitomycin C treated cell typeB) a a a a a a SI (donor) 275 SI (cell line) a IM03-7768 Proliferationbaseline of receiver 84 242 208 178.0 83.16 46.7 (allogenic donor)Control of autostimulation (Mitomycin treated autologous cells) 361 617304 427.3 166.71 39.0 Cell line type B Proliferation baseline ofreceiver 126 124 143 131.0 10.44 8.0 Control of autostimulation(Mitomycin treated autologous cells) 822 1075 487 794.7 294.95 37.1Plate ID: Plate 2 IM03-7773 Proliferation baseline of receiver 908 181330 473.0 384.02 81.2 Control of autostimulation (Mitomycin C treatedautologous cells) 269 405 572 415.3 151.76 36.5 MLR allogenic donorIM03-7768 (Mitomycin C treated) 29151 28691 28315 28719.0 418.70 1.5 MLRwith cell line (Mitomycin C treated cell type B) 567 732 905 734.7169.02 23.0 SI (donor) 61 SI (cell line) 2 IM03-7774 Proliferationbaseline of receiver 893 1376 185 818.0 599.03 73.2 Control ofautostimulation (Mitomycin C treated autologous cells) 261 381 568 403.3154.71 38.4 MLR allogenic donor IM03-7768 (Mitomycin C treated) 5310142839 48283 48074.3 5134.18 10.7 MLR with cell line (Mitomycin C treatedcell type B) 515 789 294 532.7 247.97 46.6 SI (donor) 59 SI (cell line)1 IM03-7775 Proliferation baseline of receiver 1272 300 544 705.3 505.6971.7 Control of autostimulation (Mitomycin C treated autologous cells)232 199 484 305.0 155.89 51.1 MLR allogenic donor IM03-7768 (Mitomycin Ctreated) 23554 10523 28965 21014.0 9479.74 45.1 MLR with cell line(Mitomycin C treated cell type B) 768 924 563 751.7 181.05 24.1 SI(donor) 30 SI (cell line) 1 IM03-7776 Proliferation baseline of receiver1530 137 1046 904.3 707.22 78.2 Control of autostimulation (Mitomycin Ctreated autologous cells) 420 218 394 344.0 109.89 31.9 MLR allogenicdonor IM03-7768 (Mitomycin C treated) 28893 32493 34746 32044.0 2952.229.2 MLR with cell line (Mitomycin C treated cell type B) a a a a a a SI(donor) 35 SI (cell line) a

TABLE 11-3 Average stimulation index of placenta cells and an allogeneicdonor in a mixed lymphocyte reaction with six individual allogeneicreceivers. Average Stimulation Index Recipient Placenta Plate 1(receivers 1-3) 279 3 Plate 2 (receivers 4-6) 46.25 1.3

Antigen Presenting Cell Markers—Placenta.

Histograms of placenta-derived cells analyzed by flow cytometry shownegative for production of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, asnoted by fluorescence value consistent with the IgG control, indicatingthat placental cell lines lack the cell surface molecules required todirectly stimulate allogeneic PBMCs (e.g., CD4⁺ T cells).

Immuno-Modulating Markers—Placenta.

Histograms of placenta-derived cells analyzed by flow cytometry showpositive for production of PD-L2, as noted by the increased value offluorescence relative to the IgG control, and negative for production ofCD178 and HLA-G, as noted by fluorescence value consistent with the IgGcontrol.

Summary.

In the mixed lymphocyte reactions conducted with placenta-derived celllines, the average stimulation index ranged from 1.3 to 3, and that ofthe allogeneic positive controls ranged from 46.25 to 279.Placenta-derived cell lines were negative for the production of thestimulating proteins HLA-DR, HLA-DP, HLA-DQ, CD80, CD86, and B7-H2, asmeasured by flow cytometry. Placenta-derived cell lines were negativefor the production of immuno-modulating proteins HLA-G and CD178 andpositive for the production of PD-L2, as measured by flow cytometry.Allogeneic donor PBMCs contain antigen-presenting cells expressingHLA-DP, DR, DQ, CD80, CD86, and B7-H2, thereby allowing for thestimulation of allogeneic PBMCs (e.g., naïve CD4⁺ T cells). The absenceof antigen-presenting cell surface molecules on placenta-derived cellsrequired for the direct stimulation of allogeneic PBMCs (e.g., naïveCD4⁺ T cells) and the presence of PD-L2, an immuno-modulating protein,may account for the low stimulation index exhibited by these cells in aMLR as compared to allogeneic controls.

Example 12 Secretion of Trophic Factors by Placenta-Derived Cells

The secretion of selected trophic factors from PDCs was measured.Factors were selected that have angiogenic activity (i.e., hepatocytegrowth factor (HGF) (Rosen et al. (1997) Ciba Found. Symp. 212:215-26),monocyte chemotactic protein 1 (MCP-1) (Salcedo et al. (2000) Blood 96;34-40), interleukin-8 (IL-8) (Li et al. (2003) J. Immunol. 170:3369-76),keratinocyte growth factor (KGF), basic fibroblast growth factor (bFGF),vascular endothelial growth factor (VEGF) (Hughes et al. (2004) Ann.Thorac. Surg. 77:812-8), tissue inhibitor of matrix metalloproteinase 1(TIMP1), angiopoietin 2 (ANG2), platelet derived growth factor(PDGF-bb), thrombopoietin (TPO), heparin-binding epidermal growth factor(HB-EGF), stromal-derived factor 1alpha (SDF-1alpha)),neurotrophic/neuroprotective activity (brain-derived neurotrophic factor(BDNF) (Cheng et al. (2003) Dev. Biol. 258; 319-33), interleukin-6(IL-6), granulocyte chemotactic protein-2 (GCP-2), transforming growthfactor beta2 (TGFbeta2)), or chemokine activity (macrophage inflammatoryprotein 1alpha (MIP1a), macrophage inflammatory protein 1beta (MIP1b),monocyte chemoattractant-1 (MCP-1), Rantes (regulated on activation,normal T cell expressed and secreted), 1309, thymus andactivation-regulated chemokine (TARC), Eotaxin, macrophage-derivedchemokine (MDC), IL-8).

Methods & Materials

Cell culture. PDCs derived from placenta and human fibroblasts derivedfrom human neonatal foreskin were cultured in Growth Medium (DMEM-lowglucose (Gibco, Carlsbad, Calif.), 15% (v/v) fetal bovine serum(SH30070.03; Hyclone, Logan, Utah), 50 Units/milliliter penicillin, 50microgram/milliliter streptomycin (Gibco)) on gelatin-coated T75 flasks.Cells were cryopreserved at passage 11 and stored in liquid nitrogen.After thawing of the cells, Growth Medium was added to the cellsfollowed by transfer to a 15 milliliter centrifuge tube andcentrifugation of the cells at 150×g for 5 minutes. The supernatant wasdiscarded. The cell pellet was resuspended in 4 milliliters GrowthMedium, and cells were counted. Cells were seeded at 5,000 cells/cm² ona T75 flask containing 15 milliliters of Growth Medium and cultured for24 hours. The medium was changed to a serum-free medium (DMEM-lowglucose (Gibco), 0.1% (w/v) bovine serum albumin (Sigma), 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin(Gibco)) for 8 hours. Conditioned serum-free media was collected at theend of incubation by centrifugation at 14,000×g for 5 minutes and storedat −20° C. To estimate the number of cells in each flask, cells werewashed with phosphate-buffered saline (PBS) and detached using 2milliliters trypsin/EDTA (Gibco). Trypsin activity was inhibited byaddition of 8 milliliters Growth Medium. Cells were centrifuged at 150×gfor 5 minutes. Supernatant was removed, and cells were resuspended in 1milliliter Growth Medium. Cell number was estimated using ahemocytometer.

ELISA Assay.

Cells were grown at 37° C. in 5% carbon dioxide and atmospheric oxygen.PDCs (isolate 3) also were grown in 5% oxygen or beta-mercaptoethanol(BME). The amount of MCP-1, IL-6, VEGF, SDF-1alpha, GCP-2, IL-8, andTGF-beta2 produced by each cell sample was measured by an ELISA assay(R&D Systems, Minneapolis, Minn.). All assays were performed accordingto the manufacturer's instructions. Values presented arepicogram/milliliter/million cells (n=2, sem).

SEARCHLIGHT Multiplexed ELISA Assay.

Chemokines (MIP1a, MIP1b, MCP-1, Rantes, 1309, TARC, Eotaxin, MDC, IL8),BDNF, and angiogenic factors (HGF, KGF, bFGF, VEGF, TIMP1, ANG2,PDGF-bb, TPO, HB-EGF were measured using SEARCHLIGHT Proteome Arrays(Pierce Biotechnology Inc.). The Proteome Arrays are multiplexedsandwich ELISAs for the quantitative measurement of two to 16 proteinsper well. The arrays are produced by spotting a 2×2, 3×3, or 4×4 patternof four to 16 different capture antibodies into each well of a 96-wellplate. Following a sandwich ELISA procedure, the entire plate is imagedto capture chemiluminescent signal generated at each spot within eachwell of the plate. The amount of signal generated in each spot isproportional to the amount of target protein in the original standard orsample.

Results

ELISA Assay.

MCP-1 and IL-6 were secreted by placenta-derived PDCs and dermalfibroblasts (Table 12-1). SDF-1alpha was secreted by placenta-derivedcells cultured in 5% O₂ and by fibroblasts. GCP-2 and IL-8 were secretedby placenta-derived cells cultured in the presence of BME or 5% O₂.GCP-2 also was secreted by human fibroblasts. TGF-beta2 was notdetectable by ELISA assay.

TABLE 12-1 ELISA assay results MCP-1 IL-6 VEGF SDF-1alpha GCP-2 IL-8TGF-beta2 Fibroblast 17 ± 1 61 ± 3 29 ± 2 19 ± 1 21 ± 1 ND ND Placenta -isolate 1 60 ± 3 41 ± 2 ND ND ND ND ND Placenta - isolate 2 125 ± 16 10± 1 ND ND ND ND ND Placenta - isolate 3, + BME  21 ± 10 67 ± 3 ND ND 44± 9 17 ± 9 ND Placenta - isolate 3, + 5%  77 ± 16 339 ± 21 ND 1149 ± 13754 ± 2 265 ± 10 ND O₂, W/O BME Key: ND: Not Detected.

SEARCHLIGHT Multiplexed ELISA Assay.

TIMP1, TPO, KGF, HGF, HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin,and IL-8 were secreted from placenta-derived cells (Tables 12-2 and12-3). No Ang2, VEGF, or PDGF-bb were detected.

TABLE 12-2 SEARCHLIGHT Multiplexed ELISA assay results TIMP1 ANG2 PDGFbbTPO KGF HGF FGF VEGF HBEGF BDNF HFB 19306.3 ND ND 230.5 5.0 ND ND 27.91.3 ND P1 24299.5 ND ND 546.6 8.8 16.4 ND ND 3.8 ND P3 14176.8 ND ND568.7 5.2 10.2 ND ND 1.9 33.6 Key: hFB (human fibroblasts), P1(placenta-derived cells - isolate 1), P3 (placenta-derived cells 0isolate 3). ND: Not Detected.

TABLE 12-3 SEARCHLIGHT Multiplexed ELISA assay results MIP1a MIP1b MCP1RANTES I309 TARC Eotaxin MDC IL8 HFB ND ND 39.6 ND ND 0.1 ND ND 204.9 P179.5 ND 228.4 4.1 ND 3.8 12.2 ND 413.5 P3 ND ND 102.7 ND ND 0.4 ND ND63.8 Key: hFB (human fibroblasts), P1 (placenta-derived cells - isolate1), P3 (placenta-derived cells - isolate 3). ND: Not Detected.

Summary.

Placenta-derived cells secreted a number of trophic factors. Some ofthese trophic factors, such as HGF, MCP-1, and IL-8, play importantroles in angiogenesis. Other trophic factors, such as BDNF and IL-6,have important roles in neural regeneration.

Example 13 Plasma Clotting Assay

Cell therapy may be injected systemically for certain applications wherecells are able to target the site of action. It is important thatinjected cells not cause thrombosis, which may be fatal. Tissue factor,a membrane-bound procoagulant glycoprotein, is the initiator of theextrinsic clotting cascade, which is the predominant coagulation pathwayin vivo. Tissue factor also plays an important role in embryonic vesselformation, for example, in the formation of the primitive vascular wall(Brodsky et al. (2002) Exp. Nephrol. 10:299-306). To determine thepotential for PPDCs to initiate clotting, placenta-derived PPDCs wereevaluated for tissue factor production and their ability to initiateplasma clotting.

Methods & Materials

Human Tissue Factor.

Human tissue factor SIMPLASTIN (Organon Tekailca Corporation, Durham,N.C.), was reconstituted with 20 milliliters distilled water. The stocksolution was serially diluted (1:2) in eight tubes. Normal human plasma(George King Bio-Medical, Overland Park, Kans.) was thawed at 37° C. ina water bath and then stored in ice before use. To each well of a96-well plate was added 100 microliters phosphate buffered saline (PBS),10 microliters diluted SIMPLASTIN (except a blank well), 30 microliters0.1 Molar calcium chloride, and 100 microliters of normal human plasma.The plate was immediately placed in a temperature-controlled microplatereader and absorbance measured at 405 nanometer at 40 second intervalsfor 30 minutes.

J-82 and Placenta-Derived Cells.

J-82 cells (ATCC, MD) were grown in Iscove's modified Dulbecco's medium(IMDM; Gibco, Carlsbad, Calif.) containing 10% (v/v) fetal bovine serum(FBS; Hyclone, Logan Utah), 1 milliMolar sodium pyruvate (SigmaChemical, St. Louis, Mo.), 2 milliMolar L-Glutamin (Mediatech Herndon,Va.), 1×non-essential amino acids (Mediatech Herndon, Va.). At about 70%confluence, cells were transferred to wells of 96-well plate at 100,000,50,000 and 25,000 cells/well. Placenta-derived cells were cultured inGrowth Medium (DMEM-low glucose (Gibco), 15% (v/v) FBS, 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin(Gibco), and 0.001% betamercaptoethanol (Sigma)) in gelatin-coated T75flasks (Corning, Corning, N.Y.). Placenta-derived cells at passage 5were transferred to wells at 50,000 cells/well. Culture medium wasremoved from each well after centrifugation at 150×g for 5 minutes.Cells were suspended in PBS without calcium and magnesium.

Tissue Factor Inhibition.

Inhibition of the clotting reaction by preincubation of cells with CNTO859, an antibody to tissue factor, will demonstrate that tissue factoris responsible for the clotting. Cells are incubated with 20microgram/milliliter CNTO 859 (Centocor, Malvern, Pa.) for 30 minutes.Calcium chloride (30 microliter) is added to each well. The plate isimmediately placed in a temperature-controlled microplate reader andabsorbance measured at 405 nanometer at 40 second intervals for 30minutes. Cells are washed in PBS and detached from the flask withTrypsin/EDTA (Gibco Carlsbad, Calif.). Cells are harvested, centrifuged,and re-suspended 3% (v/v) FBS in PBS at a cell concentration of 1×10⁷per milliliter. Antibody is added to 100 microliter cell suspension asper the manufacturer's specifications, and the cells are incubated inthe dark for 30 minutes at 4° C. After incubation, cells are washed withPBS and centrifuged at 150×g for 5 minutes to remove unbound antibody.Cells are re-suspended in 100 microliter of 3% FBS and secondaryantibody added as per the manufacturer's instructions. Cells areincubated in the dark for 30 minutes at 4° C. After incubation, cellsare washed with PBS and centrifuged to remove unbound secondaryantibody. Washed cells are re-suspended in 500 microliter of PBS andanalyzed by flow cytometry.

Results

Flow cytometry analysis revealed that placenta-derived postpartum cellsexpress tissue factor. Placenta-derived cells increased the clottingrate as indicated by the time to half maximal absorbance (T ½ to max;Table 13-1). The T ½ to max is inversely proportional to the number ofJ82 cells.

TABLE 13-1 The effect of human tissue factor (SIMPLASTIN) and placenta-derived cells (Pla) on plasma clotting was evaluated. The time to halfmaximal absorbance (T ½ to max) at the plateau in seconds was used as ameasurement unit. SIMPLASTIN Dilution T ½ to max (seconds) 1:2 61 1:4107 1:8 147  1:16 174  1:32 266  1:64 317  1:128 378 0 (negativecontrol) 1188 J-82 cells 100,000  122 50,000 172 25,000 275 Pla P550,000 757

Summary.

Placenta-derived cells express tissue factor. Tissue factor is normallyfound on cells in a conformation that is inactive but is activated bymechanical or chemical (e.g., LPS) stress (Sakariassen et al. (2001)Thromb. Res. 104:149-74; Engstad et al. (2002) Int. Immunopharmacol.2:1585-97). Thus, minimization of stress during the preparation processof PDCs may prevent activation of tissue factor. In addition to thethrombogenic activity, tissue factor has been associated with angiogenicactivity. Thus, tissue factor activity may be beneficial whenplacenta-derived cells are transplanted in tissue but should beinhibited when PDCs are injected intravenously.

Example 14 Differentiation of Placenta-Derived Cells into Hepatocytes

A variety of conditions were examined to determine a suitablecombination of basic media and growth factors for the differentiation ofplacenta-derived cells into hepatocytes. HNF-1alpha, ahepatocyte-specific transcription factor, cytoplasmic intermediatefilament proteins such as keratin 19 (K19), keratin 8 (K8), andcytokeratin 18 (CK18), which are markers of epithelial cells and twoliver-specific secreted proteins, alpha-fetoprotein (alphaFP), andalbumin were selected as markers for hepatocyte differentiation(Schwartz et al. (2002) J. Clin. Invest. 109(10):1291-1302; Okumoto etal. (2003) Biochem. Biophys. Res. Commun. 304(4):691-695; Chagraoui etal. (2003) Blood 101(8): 2973-2982).

Methods & Materials

Placenta-derived cells isolated according to the method described inExample 1, as well as neonatal or adult Normal Human Dermal Fibroblasts(NHDF) were grown in Growth medium (DMEM-low glucose (Gibco, Carlsbad,Calif.), 15% (v/v) fetal bovine serum (Cat. #SH30070.03; Hyclone, LoganUtah), 0.001% (v/v) beta-mercaptoethanol (Sigma, St. Louis, Mo.), 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin(Gibco)), in a gelatin-coated T75 flask. Basic Fibroblast Growth Factor(bFGF), Oncostatin M, Hepatocyte Growth Factor (HGF), Stem Cell Factor(SCF), and Fibroblast Growth Factor 4 (FGF 4) were from PeproTech Inc.(Rocky Hill, N.J.). Platelet Derived Growth Factor BB (PDGF-BB) was fromR&D Systems (Minneapolis, Minn.).

The following conditions were tested:

Method 1

Placenta-Derived Cells (P2) (Predominately Neonatal as Analyzed byKaryotyping), Neonatal and Adult Normal Human Dermal Fibroblasts (NHDF).

Cells were plated at 22.5×10³ cells/cm² on 1% MATRIGEL (BD DiscoveryLabware, Bedford, Mass.) (Becton-Dickinson and Co., Franklin Lakes,N.J.) in serum-free medium (60% (v/v) low glucose DMEM) (DMEM-LG; Gibco,Carlsbad, Calif.), 40% (v/v) MCDB-201 (Sigma, St. Louis, Mo.),supplemented with 1× insulin/transferrin/selenium, 4.7microgram/milliliter linoleic acid, 1 milligram/milliliter bovine serumalbumin, 10 nanoMolar Dexamethasone, 100 microMolar ascorbic acidphosphate (all from Sigma), 100 Units/milliliter penicillin, 100Units/milliliter streptomycin (Gibco), 2% (v/v) FCS (HycloneLaboratories, Logan, Utah), and 10 nanogram/milliliter each EGF andPDGF-BB). After 8 to 12 hours, medium was removed, cells were washedtwice with PBS (Gibco) and cultured in the above-described mediumwithout EGF and PDGF-BB but supplemented with 20 nanogram/milliliter HGFand/or 10 nanogram/milliliter FGF-4 (Schwartz et al. (2002) J. Clin.Invest. 109(10):1291-1302). Cells were cultured in standard air with 5%CO₂ at 37° C.

Method 2

Placenta-Derived Cells (P2) (Predominately Neonatal as Analyzed byKaryotyping), Neonatal and Adult NHDF.

Cells were seeded at 22,500 cells/cm² in 24-well plates coated withgelatin and grown as described above.

Method 3

Placenta-Derived Cells (P10), Adult NHDF, Placenta-Derived Cells (P3).

Cells were seeded at high density (50,000 cells/cm²) in 24-well TCPplates and grown in DMEM (Gibco), B27 Supplement (Gibco), 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin, 20nanograms/milliliter HGF and/or 10 nanograms/milliliter FGF-4. Cellswere grown in these conditions for 4 weeks.

Method 4

Placenta-Derived Cells (P3), Placenta-Derived Cells (P15),Placenta-Derived Cells (P2) (Predominately Neonatal as Analyzed byKaryotyping), Placenta-Derived Cells (P5) (Predominately Neonatal asAnalyzed by Karyotyping), Placenta-Derived Cells (P5) (PredominatelyMaternal as Analyzed by Karyotyping), Neonatal and Adult NHDF.

Cells were seeded at a density of 5,000 cells/cm² in T25 flasks in ChangC medium (Irvine Scientific, Santa Ana, Calif.) on either fibronectin(PeproTech, Rocky Hill, N.J.) or gelatin (Sigma) and grown for twopassages until confluence. Cells were then seeded at 1,000 cells/cm² in24-well TCPS plates and grown as described above until they reachedabout 40-60% confluence.

Method 5

Placenta-Derived Cells (P2) (Predominately Neonatal as Analyzed byKaryotyping), and Adult NHDF.

Cells were plated in 24-well plates on gelatin in Growth mediumsupplemented with either 1 nanogram/milliliter or 10 nanogram/milliliteroncostatin M (Chagraoui (2003) Blood 101(8): 2973-2982). Cells weregrown in these conditions for 4 weeks.

Method 6

Placenta-Derived Cells (P2) (Predominately Neonatal as Analyzed byKaryotyping), and Adult NHDF.

Cells were plated in 24-well plates on gelatin in Growth mediumsupplemented with 10 nanogram/milliliter bFGF, 10 nanogram/milliliterHGF, 10 nanogram/milliliter SCF. Cells were grown in these conditionsfor 4 weeks (Okumoto et al. (2003) Biochem. Biophys. Res. Commun.304(4):691-695.).

Total RNA Isolation and Quantitative RT-PCR.

RNA was extracted from placenta-derived cells and fibroblasts grown asdescribed in each protocol. Cells were lysed with 350 microliter bufferRLT containing beta-mercaptoethanol (Sigma St. Louis, Mo.) according tothe manufacturer's instructions (RNeasy Mini Kit, Qiagen, Valencia,Calif.) and RNA extracted according to the manufacturer's instructions(RNeasy Mini Kit, Qiagen, Valencia, Calif.) with a 2.7 Units/sampleDNase treatment (Sigma). RNA was eluted with 50 microliter DEPC-treatedwater and stored at −80° C. RNA was reverse transcribed using randomhexamers with the TaqMan reverse transcription reagents (AppliedBiosystems, Foster City, Calif.) at 25° C. for 10 minutes, 37° C. for 60minutes, and 95° C. for 10 minutes. Samples were stored at −20° C.

Real-Time PCR.

PCR was performed on cDNA samples using ASSSAYS-ON-DEMAND geneexpression products for albumin (Hs00609411), cytochrome p450 2B6(Hs00167937), GAPDH (Applied Biosystems, Foster City, Calif.) and TaqManUniversal PCR master mix according to the manufacturer's instructions(Applied Biosystems, Foster City, Calif.) using a 7000 sequencedetection system with ABI prism 7000 SDS software (Applied Biosystems,Foster City, Calif.). Thermal cycle conditions were initially 50° C. for2 min and 95° C. for 10 minute followed by 40 cycles of 95° C. for 15seconds and 60° C. for 1 minute. PCR data were analyzed according tomanufacturer's specifications (User Bulletin #2 from Applied Biosystemsfor ABI Prism 7700 Sequence Detection System).

Immunofluorescence.

Cell cultures were fixed with cold 4% (w/v) paraformaldehyde for aperiod of 10 minutes at room temperature. Immunocytochemistry wasperformed using antibodies directed against the following epitopes:keratin 8 (K8; 1:400; Chemicon, Temecula, Calif.), keratin 19 (K19;1:400; Chemicon), cytokeratin 18 (CK18; 1:400; Sigma, St. Louis, Mo.),vimentin (1:500; Sigma), desmin (1:150; Sigma), albumin (1:200; Sigma),c-met (1:400; Santa Cruz Biotech, Santa Cruz, Calif.), and HNF-1alpha(1:400; Santa Cruz Biotech). In general, cultures were washed withphosphate-buffered saline (PBS) and exposed to a protein blockingsolution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula,Calif.), and 0.3% (v/v) Triton (Triton X-100, Sigma) for 30 minutes toaccess intracellular antigens. In instances where the epitope ofinterest would be located on the cell surface (c-met), triton wasomitted in all steps of the procedure in order to prevent epitope loss.Primary antibodies, diluted in blocking solution, were then applied tothe cultures for a period of 1 hour at room temperature. Next, primaryantibody solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing blocking solution along with goat anti-mouse IgG—Texas Red(1:250; Molecular Probes, Eugene, Oreg.) for K8, K19, CK18, vimentin,and albumin, goat anti-rabbit IgG—Alexa 488 (1:250; Molecular Probes)for desmin and c-met, or donkey anti-goat IgG—FITC (1:150; Santa CruzBiotech) for HNF-1alpha staining. Cultures were washed and 10 microMolarDAPI (Molecular Probes) was applied for 10 minutes to visualize cellnuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). Representative images werecaptured using a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Results

In order to determine whether placenta-derived cells could expressepithelial markers, cells were cultured in Chang C medium.Placenta-derived cells (P4), (P3), and (P8) were grown in Chang C mediumfor 11 days. Placenta-derived cells stained positive for cytokeratin 18by immunocytochemistry analysis. None of the samples stained positivefor keratin 8. Samples grown in Growth medium were negative for bothmarkers.

The effect of early and late passages as well as gelatin and fibronectinsubstrata was investigated. Cells were grown in Chang C medium for 11days. RNA and protein expression of epithelial/hepatocyte-specificproteins were analyzed. Immunocytochemistry staining for cytokeratin18,keratin 8, keratin 19, c-met, albumin, desmin, and HNF-1alpha werenegative in all conditions. Cells stained positive for vimentin.Expression of both albumin and cytochrome p450 2B6 at levels lower thanthat of human HepG2 cells was detected with assay-on-demand primers.Albumin and cytochrome p450 2B6 expression also were detected in cellsgrown in Growth medium.

Placenta-derived cells were treated as described in method 1 accordingto a protocol developed by Schwartz et al. (2002) J. Clin. Invest.109(10):1291-1302.). Both albumin and cytochrome p450 2B6 were detectedwith assay-on-demand primers at levels lower than HepG2 positivecontrol. No clear pattern emerged between conditions applied and geneexpression levels, i.e., albumin and cytochrome p450 2B6 expression wasalso detected in control samples. Some expression of albumin andcytochrome p450 2B6 was detected with ASSAY-ON-DEMAND primers howeverthe levels were significantly lower than those observed in human HepG2cells.

Oncostatin M at low concentration of 1 nanogram/milliliter increasedexpression levels of cytochrome p450 2B6 in placenta-derived cells grownin Growth medium on gelatin-coated flasks. FGF-4 and HGF treatment hadlittle effect and may have reduced the expression of albumin andcytochrome p450 2B6.

Summary.

Several differentiation protocols were tested for ability to inducedifferentiation of placenta-derived cells to hepatocyte phenotype.Expression of hepatocyte-specific markers such as albumin and cytochromep450 2B6 was detected, thereby indicating that the cells underwent somedifferentiation into hepatocytes. Placenta-derived cells cultured inChang C medium expressed cytokeratin 18, a marker of epithelial cells inthe lower or pancreatic ducts.

Example 15 Differentiation of Placenta-Derived Cells to an OsteogenicPhenotype

Mesenchymal stem cells (MSCs) derived from bone marrow have beendemonstrated to reproducibly differentiate into osteoblast-like cellsthat mineralize and express alkaline phosphatase. Additional markersexpressed by osteoblasts, such as osteocalcin and bone sialoprotein,have been used to demonstrate differentiation into an osteoblast-likecell. The ability of placenta-derived cells to differentiate into anosteogenic phenotype was evaluated by culturing in an osteogenic mediumand addition of bone morphogenic proteins (BMP)-2 (Rickard et al. (1994)Dev. Biol. 161:218-228) or 4 and transforming growth factor beta1.

Methods & Materials

Culture of Cells.

Prior to initiation of osteogenesis, Mesenchymal Stem Cells (MSC) weregrown in Mesenchymal Stem Cell Growth Medium Bullet kit (MSCGM, Cambrex,Walkerville, Md.). Other cells were cultured in Growth medium (DMEM-lowglucose (Gibco, Carlsbad, Calif.), 15% (v/v) fetal bovine serum(SH30070.03; Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol(Sigma, St. Louis, Mo.), 50 Units/milliliter penicillin, 50microgram/milliliter streptomycin (Gibco)) in a gelatin-coated T75 flaskand were washed with phosphate buffered saline (PBS).

Osteoblasts (9F1721; Cambrex) were grown in osteoblast growth medium(Cambrex) and RNA was extracted as described below.

Osteogenesis

Protocol 1.

Placenta-derived cells (P3) and (P4) (previously karyotyped and shown tobe predominantly neonatal-derived cells) and MSCs (P3) were seeded at5×10³ cells/cm² in 24-well plates and 6-well dishes in Growth medium andincubated overnight. The medium was removed and replaced with Osteogenicmedium (DMEM-low glucose, 10% (v/v) fetal bovine serum, 10 milliMolarbetaglycerophosphate (Sigma), 100 nanoMolar dexamethasone (Sigma, St.Louis, Mo.), 50 microMolar ascorbate phosphate salt (Sigma), fungizone(Gibco), 50 Units/milliliter penicillin, 50 microgram/milliliterstreptomycin (Gibco)). Osteogenic medium was supplemented with 20nanogram/milliliter TGF-beta1 (Sigma), 40 nanogram/milliliter hrBMP-2(Sigma) or 40 nanogram/milliliter hrBMP-4 (Sigma). Cultures were treatedfor a total of 14, 21, and 28 days, with media changes every 3-4 days.

Protocol 2.

Placenta-derived cells were tested for the ability to differentiate intoan osteogenic phenotype. Placenta-derived cells (P4) were seeded at30,000 cells/well of a 6-well plate (gelatin-coated) in Growth medium.Mesenchymal stem cells (MSC) (P3 and P4), fibroblasts (P11), and iliaccrest bone marrow cells (P3; International PCT Publication No.WO03/025149) were seeded at 30,000 cells/well of a 6 well plate(gelatin-coated) in mesenchymal stem cell growth medium (MSCGM, Cambrex)and Growth medium, respectively.

Osteogenic induction was initiated by removing the initial seeding media(24 h) and replacing it with osteogenic induction medium: DMEM-lowglucose, 10% fetal bovine serum, 10 millimolar betaglycerophosphate(Sigma), 100 nanoMolar dexamethasone (Sigma), 50 microMolar ascorbatephosphate salt (Sigma), 50 Units/milliliter penicillin, 50microgram/milliliter streptomycin (Gibco). In some conditions,osteogenic medium was supplemented with human recombinant (hr) BMP-2 (20nanogram/milliliter) (Sigma,) or hrBMP-4 or with both hrBMP-2 (20nanogram/milliliter) and hrBMP-4 (20 nanogram/milliliter) (Sigma).Cultures were treated for a total of 28 days with media changes every3-4 days.

RNA Extraction and Reverse Transcription.

Cells were lysed with 350 microliter buffer RLT containingbetamercaptoethanol (Sigma, St. Louis, Mo.) according to themanufacturer's instructions (RNeasy Mini kit, Qiagen, Valencia, Calif.)and stored at −80° C. Cell lysates were thawed and RNA extractedaccording to the manufacturer's instructions (RNeasy Mini kit, Qiagen,Valencia, Calif.) with a 2.7 Unit/sample DNase treatment (Sigma St.Louis, Mo.). RNA was eluted with 50 micoliter DEPC-treated water andstored at −80° C. RNA was reverse transcribed using random hexamers withthe TaqMan reverse transcription reagents (Applied Biosystems, FosterCity, Calif.) at 25° C. for 10 minutes, 37° C. for 60 minutes, and 95°C. for 10 minutes. Samples were stored at −20° C.

PCR.

PCR was performed on cDNA samples using ASSAYS-ON-DEMAND gene expressionproducts bone sialoprotein (Hs00173720), osteocalcin (Hs00609452), GAPDH(Applied Biosystems, Foster City, Calif.), and TaqMan Universal PCRmaster mix according to the manufacturer's instructions (AppliedBiosystems, Foster City, Calif.) using a 7000 sequence detection systemwith ABI prism 7000 SDS software (Applied Biosystems, Foster City,Calif.). Thermal cycle conditions were initially 50° C. for 2 minutesand 95° C. for 10 minutes followed by 40 cycles of 95° C. for 15 secondsand 60° C. for 1 minute.

von Kossa Staining.

Cells were fixed with 10% (v/v) neutral buffered formalin(Richard-Allan, Kalamazoo, Mich.). After fixation, the cells were washedin deionized water and incubated in 5% (w/v) silver nitrate (AldrichChemical Company Milwaukee, Wis.) for one hour in direct sunlight. Cellswere washed in deionized water and incubated in 5% (w/v) sodiumthiosulfate (EM Sciences, Gibbstown, N.J.) for five minutes. Cells werethen washed in distilled water and examined by light microscopy.

Results

Protocol 1.

RNA extracted from osteoblasts was used as a positive control for thereal-time gene expression of osteocalcin and bone sialoprotein.Osteoblast expression levels of osteocalcin and BSP relative toplacenta-derived cells grown in growth medium were 2.5- and 8000-fold,respectively. MSCs grown in the osteogenic medium mineralized and gavepositive von Kossa staining. MSC expression of osteocalcin and BSP wassignificantly increased in osteogenic medium at 21 days. The addition ofBMP-2 and -4 enhanced BSP expression but had no effect on osteocalcinexpression. TGF-beta1 did not augment the effect of osteogenesis medium.Extensive mineralization was observed with one placenta sample (P4) thathad predominantly neonatal-derived cells. Placenta-derived cells (P3)showed induction of BSP expression levels in osteogenic media and lowlevels of osteocalcin induction. BMP-4 and TGF-beta1 increasedosteocalcin expression by placenta-derived cells (P3).

Protocol 2.

Osteogenic differentiation, as shown by positive von Kossa staining formineralization, was observed with placenta-derived cells (P4) and ICBM(P3) incubated with osteogenic medium supplemented with BMP2 or 4, andMSCs (P3) incubated with osteogenic medium supplemented with BMP 4(Table 15-1). None of the other cells differentiated into the osteogenicphenotype and stained by von Kossa. To ensure that von Kossa stainingwas related to the cell and not extracellular matrix, cells werecounterstained with nuclear fast red. This stain demonstrated largelipid droplets in some MSCs consistent with an adipocyte phenotype. Thissuggests that MSCs do not differentiate specifically into an osteogenicphenotype in these conditions. Furthermore, the level of adipogenesiswas seen to increase when MSCs were incubated in osteogenic mediumsupplemented with either BMP2 or BMP4.

TABLE 15-1 Results of osteogenic differentiation using von Kossastaining for Protocol 2. Placenta-derived cells (Pla), mesenchymal stemcells (MSC), fibroblasts (Fib), and iliac crest bone marrow cells (ICBM)cells were cultured in osteogenic medium (OM) alone or supplemented withBMP2 or BMP2 and BMP4. Von Number Cell Line Conditions Kossa Comments 1ICBM P3 Osteogenic Neg Normal O2 medium (OM) 2 ICBM P3 OM, BMP2 PosNormal O3 3 ICBM P3 OM, BMP4 Pos Normal O4 4 MSC Osteogenic Neg lots offat medium (OM) 5 MSC OM, BMP2 Neg lots of fat 6 MSC OM, BMP4 Pos lotsof fat 7 Pla P4 Osteogenic Neg medium (OM) 8 Pla P4 OM, BMP2 Pos 9 PlaP4 OM, BMP4 Pos 10 MSC P4 Osteogenic Neg Fat medium (OM) 11 MSC P4 OM,BMP2 Neg Fat 12 MSC P4 OM, BMP2, BMP4 Neg Fat 13 Pla P4 Osteogenic Negmedium (OM) 14 Pla P4 OM, BMP2 Neg 15 Pla P4 OM, BMP2, BMP4 Neg 16 Fib1F1853 P11 Osteogenic Neg medium (OM) 17 Fib 1F1853 P11 OM, BMP2 Neg 18Fib 1F1853 P11 OM, BMP2, BMP4 Neg

Summary.

Bone marrow-derived MSCs (Kadiyala et al. (1997) Cell Transplant.6:125-34) as well as cells derived from other tissue such as adipose(Halvorsen et al. (2001) Tissue Eng. 7:729-41) have been shown todifferentiate into an osteoblast-like cell. MSCs have also been shown todifferentiate into adipocytes or osteoblasts in response to BMPs (Chenet al. (1998) J. Cell Biol. 142:295-305) due to differential roles forbone morphogenic protein (BMP) receptor type IB and IA. Placenta-derivedcells are also capable of expressing an osteoblast-like phenotype aspreviously observed with bone marrow-derived mesenchymal stem cells(MSCs) when placed in osteogenic medium containing dexamethasone,B-glycerophosphate, and ascorbic acid. Several experiments wereconducted with different isolates to determine whether there wasmineralization of the cultured cells by von Kossa staining andexpression of bone sialoprotein (BSP) and osteocalcin, which areexpressed in osteoblasts. Following induction of osteogenesis, MSCs weredemonstrated to mineralize and stain with von Kossa and also haveincreased mRNA levels of bone sialoprotein and osteocalcin expressionusing real-time relative quantitation. Numerous MSCs also formed lipiddroplets in the cytoplasm similar to adipocytes. Placenta-derived cells(predominantly neonatal cells) showed extensive mineralization andinduction of BSP and osteocalcin in osteogenic medium, which wasenhanced at 21 days with BMP-2 or -4.

Example 16 Chondrogenic Differentiation of Placenta-Derived Cells

Placenta-derived cells were tested for their ability to differentiateinto chondrocytes in vitro in two different assay systems: the pelletassay culture system and collagen gel cultures. The pellet culturesystem has been used successfully with selected lots of humanmesenchymal stem cells (MSC). MSC grown in this assay and treated withtransforming growth factor-beta3 have been shown to differentiate intochondrocytes (Johnstone, et al. (1998) Exp. Cell Res. 238:265-272). Thecollagen gel system has been used to culture chondrocytes in vitro(Gosiewska, et al. (2001) Tissue Eng. 7:267-277.). Chondrocytes grownunder these conditions form a cartilage-like structure.

Materials and Methods

Cell Culture

Human placentas were received and cells were isolated as described(Example 1). Cells were cultured in Growth medium (Dulbecco's ModifiedEssential Media (DMEM), 15% (v/v) fetal bovine serum (Hyclone, LoganUtah), 50 Units/milliliter penicillin, 50 microgram/milliliterstreptomycin (Invitrogen, Carlsbad, Calif.), 0.001% (v/v)2-mercaptoethanol (Sigma, St. Louis, Mo.)) on gelatin-coated tissueculture plastic flasks. The cultures were incubated at 37° C. with 5%CO₂. For use in experiments, cells were between passages 4 and 12.

Human articular chondrocytes were purchased from Cambrex (Walkersville,Md.) and cultured in the same media as the placenta-derived cells.Twenty-four hours before the experiment, the culture media was changedto a media containing 1% FBS.

Human mesenchymal stem cells (MSCs) were purchased from Cambrex(Walkersville, Md.) and cultured in MSCGM (Cambrex). Cells used forexperiments were between passages 2 and 4.

Collagen Gel Assays.

Cultured cells were trypsinized to remove from culture plate. Cells werewashed with centrifugation twice at 300×g for 5 min in DMEM withoutserum and counted. Cells were mixed with the following components at thefinal concentrations listed: rat tail collagen (1 milligram/milliliter,BD DiscoveryLabware, Bedford, Mass.), 0.01 Normal NaOH, and Chondrogenicmedium (DMEM, 100 Units/milliliter penicillin, 100 microgram/milliliterstreptomycin, 2 millimolar L-Glutamine, 1 millimolar Sodium Pyruvate,0.35 millimolar L-Proline, 100 nanoMolar dexamethasone, 0.17 millimolarL-Ascorbic Acid, 1% (v/v) ITS (insulin, transferrin, selenium) (allcomponents from Sigma Chemical Company)). The cells were gently mixedwith the medium, and the samples were aliquoted into individual wells ofa 24-well ultra-low cluster plate (Corning, Corning, N.Y.) at aconcentration of either 2×10⁵ per well or 5×10⁵ per well. Cultures wereplaced in an incubator and left undisturbed for 24 to 48 hours. Mediumwas replaced with fresh chondrogenic medium supplemented withappropriate growth factor every 24-48 hours. Samples were allowed toculture for up to 28 days at which time they were removed and fixed in10% (v/v) formalin (VWR Scientific, West Chester, Pa.) and processed forhistological examination. Samples were stained with Safranin O orhematoxylin/eosin for evaluation.

Pellet Culture Assays.

Cultured cells were trypsinized to remove from the culture plate. Cellswere washed with centrifugation twice at 300×g for 5 minutes in DMEMwithout serum and counted. Cells were resuspended in fresh chondrogenicmedium (described above) at a concentration of 5×10⁵ cells permilliliter. Cells were aliquoted into new polypropylene tubes at 2.5×10⁵cells per tube. The appropriate samples were then treated with eitherTGF-beta3 (10 nanogram/milliliter, Sigma) or GDF-5 (100nanogram/milliliter; R&D Systems, Minneapolis, Minn.) as growth factor.Cells were then centrifuged at 150×g for 3 minutes. Tubes were thentransferred to the incubator and left undisturbed for 24 to 48 hours instandard atmosphere with 5% CO₂ at 37° C. and. Media was replaced withfresh chondrocyte cell media and growth factor, where appropriate, every2 to 3 days. Samples were allowed to culture for up to 28 days at whichtime they were removed and fixed and stained as described above.

Results

Safranin O stains of cell pellets of placenta-derived cells treated withTGF-beta3 and GDF-5 showed positive Safranin O staining as compared tocontrol cells, indicating glycosoaminoglycan. Placenta-derived cellsalso showed some chondrocyte-like morphology.

Summary.

The results of the present study show that the placenta-derived cellspartially differentiated into chondrocytes in vitro in the pelletculture and the collagen gel assay systems, as evidenced byglycosaminoglycan expression and similarity of cell morphology tocartilage tissue.

Example 17 Evaluation of Chondrogenic Potential of Placenta-DerivedCells in an In Vitro Pellet Culture Based Assay

This example describes evaluation of the chondrogenic potential of cellsderived from placental tissue using in vitro pellet culture basedassays. Cells derived from placenta at early passage (P3) and latepassage (P12) were used. The chondrogenic potential of the cells wasassessed in pellet culture assays, under chondrogenic inductionconditions, in medium supplemented with transforming growth factorbeta-3 (TGF beta-3), rhGDF-5 (recombinant human growth anddifferentiation factor 5) or a combination of both.

Materials & Methods

Reagents.

Dulbecco's Modified Essential Media (DMEM), Penicillin and Streptomycin,were obtained from Invitrogen, Carlsbad, Calif. Fetal calf serum (FCS)was obtained from HyClone (Logan, Utah). Mesenchymal stem cell growthmedium (MSCGM) and hMSC chondrogenic differentiation bullet kit wereobtained from Biowhittaker, Walkersville, Md. TGF beta-3 was obtainedfrom Oncogene research products, San Diego, Calif. rhGDF-5 was obtainedfrom Biopharm, Heidelberg, Germany (WO9601316 A1, U.S. Pat. No.5,994,094 A).

Cells.

Human mesenchymal stem cells (Lot#2F1656) were obtained fromBiowhittaker, Walkersville, Md. and were cultured in MSCGM according tomanufacturer's instructions. This lot has been tested previously, andwas shown to be positive in the chondrogenesis assays. Human adult andneonatal fibroblasts were obtained from American Type Culture Collection(ATCC), Manassas, Va. and cultured in growth medium (Dulbecco's ModifiedEssential supplemented with 15% (v/v) fetal bovine serum, 100Units/milliliter penicillin, 100 microgram/milliliter streptomycin and0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.) on gelatin-coatedtissue culture plastic flasks. Placenta-derived cells (Lot#071003Plac)were utilized. Cells were cultured in Growth medium similar tofibroblasts. The cell cultures were incubated at 37° C. with 5% CO₂.Cells used for experiments were at passages 3 and 12.

Pellet Culture Assay.

For pellet cultures, 0.25×10⁶ cells were placed in a 15 milliliterconical tube and centrifuged at 150×g for 5 minutes at room temperatureto form a spherical pellet according to protocol for chondrogenic assayfrom Biowhittaker. Pellets were cultured in chondrogenic inductionmedium containing TGF beta-3 (10 nanogram/milliliter), rhGDF-5 (500nanogram/milliliter), or a combination of TGF beta-3 (10nanogram/milliliter), and rhGDF-5 (500 ng/ml) for three weeks. Untreatedcontrols were cultured in growth medium. During culture, pellets werere-fed with fresh medium every other day. Treatment groups included thefollowing:

Treatment Group

A. Placenta-derived cells early passage (P EP)+rhGDF-5

B. Placenta-derived cells late passage (P LP)+rhGDF-5

C. Human Mesenchymal Stem cells (HMSC)+rhGDF-5

D. Human adult fibroblast cells (HAF)+rhGDF-5

E. Placenta-derived cells early passage (P EP)+TGF beta-3

F. Placenta-derived cells late passage (P LP)+TGF beta-3

G. Human Mesenchymal Stem cells (HMSC)+TGF beta-3

J. Human adult fibroblast cells (HAF)+TGF beta-3

I. Placenta-derived cells early passage (P EP)+rhGDF-5+TGF beta-3, n=1

J. Placenta-derived cells late passage (P LP)+rhGDF-5+TGF beta-3

K. Human Mesenchymal Stem cells (HMSC)+rhGDF-5+TGF beta-3

L. Human adult fibroblast cells (HAF)+rhGDF-5+TGF beta-3

M. Human neonatal fibroblast cells (HNF)+rhGDF-5+TGF beta-3

N. Placenta-derived cells early passage (P EP)

O. Placenta-derived cells late passage (P LP)

P. Human Mesenchymal Stem cells (HMSC)

Q. Human adult fibroblast cells (HAF)

Histology of In Vitro Samples.

At the end of the culture period pellets were fixed in 10% bufferedformalin and sent to MPI Research (Mattawan, Mich.) for paraffinembedding, sectioning, and staining with Hematoxylin/Eosin (H/E) andSafranin O (SO) staining.

Results

Placenta-derived cells, MSCs and fibroblasts formed cell pellets inchondrogenic induction medium with the different growth factors. Thesize of the pellets at the end of culture period varied among thedifferent cell types. Pellets formed with the placental cells weresimilar in size, or slightly larger than, those formed by MSCs andfibroblasts. Pellets formed with all cell types and cultured in controlmedium were smaller than pellets cultured in chondrogenic inductionmedium.

Examination of cross sections of pellets stained with H/E and Safranin-Oprovided some indication that placenta-derived cells at early and latepassage may have the potential to undergo chondrogenic differentiation.Chondrogenesis as assessed by cell condensation, cell morphology andSafranin O positive staining of matrix was indistinct in theplacenta-derived cells cultured in chondrogenic induction mediumsupplemented with TGF beta-3, rhGDF-5, or both. However, this may be dueto the fact that chondrogenic induction conditions were optimized forMSCs, not for postpartum-derived cells, and it should be noted thatcontrol pellets cultured in growth medium showed no evidence ofchondrogenesis. Moreover, distinct cell populations were observed inplacenta-derived cells at both passages located apically or centrally.Some cell condensation was observed with fibroblast, but it was notassociated with Safranin O staining.

Example 18 Adipogenic Differentiation of Placenta-Derived Cells

Stromal populations of stem cells have been demonstrated todifferentiate into an adipogenic phenotype (Janderova et al. (2003)Obes. Res. 11(1):65-74; Zangani et al. (1999) Differentiation64(2):91-101; Liu et al. (2003) Curr. Mol. Med. 3(4):325-40). Thepotential of placenta-derived cells to differentiate into an adipogenicphenotype was examined.

Methods & Materials

Adipose Differentiation.

Placenta-derived cells (P3) were seeded at 200,000 cells per well on6-well tissue culture-treated plates in growth medium ((DMEM:Low glucose(Invitrogen, Carlsbad, Calif.), 15 percent (v/v) defined bovine serum(Hyclone, Logan, Utah; Lot#AND18475), 0.001 percent 2-mercaptoethanol(Sigma, St. Louis, Mo.), 100 Units/milliliter penicillin, 100microgram/milliliter streptomycin, 0.25 micrograms per milliliteramphotericin B; Invitrogen, Carlsbad, Calif.). Mesenchymal stem cells(P3, IF2155), osteoblasts (P5, CC2538; Cambrex, Walkerville, Md.),omental cells (P6) (isolated from omental tissue from NDRI, followingprotocol used for placenta-derived cell isolation in Example 1),adipose-derived cells (U.S. Pat. No. 6,555,374 B1) (P6), and fibroblasts(P6, CC2509) (Cambrex, Walkerville, Md.) were also seeded under the sameconditions. Prior to initiation of osteogenesis, Mesenchymal Stem Cellswere grown in a Mesenchymal Stem Cell Growth Medium Bullet kit (Cambrex,Walkerville, Md.). After 2 days, spent medium was aspirated off andcells were washed with phosphate buffered saline (PBS). At this point,medium was switched to Dulbecco's minimal essential medium-high glucose(DMEM-Hg; Invitrogen, Carlsbad, Calif.) containing 10 percent FBS (v/v,Hyclone, Logan Utah), 0.02 milligrams per milliliter insulin (Sigma, St.Louis, Mo.), and 100 Units/milliliter penicillin, 100microgram/milliliter streptomycin, 0.25 micrograms per milliliteramphotericin B; Invitrogen, Carlsbad, Calif.). Once the cells hadreached confluence, spent medium was aspirated off. Cells were thencultured in an adipose differentiation medium (DMEM-Hg (Invitrogen,Carlsbad, Calif.), containing 10 percent defined fetal bovine serum((v/v), Hyclone, Logan, Utah), 0.02 milligrams per milliliter insulin(Sigma, St. Louis, Mo.) and 100 Units/milliliter penicillin, 100micrograms/milliliter streptomycin, and 0.25 micrograms/milliliteramphotericin, 5 micromolar isobutylmethylxanthine (Sigma, St. Louis,Mo.), 100 micromolar dexamethasone (Sigma, St. Louis, Mo.), and 2.5micromolar indomethacin (Sigma, St. Louis, Mo.) for up to 4 weeks. Cellswere stained with Oil-Red-O to determine the presence of lipid dropletformation.

Oil Red O Staining.

Cells were fixed with 10 percent (v/v) neutral buffered formalin(Richard-Allan Kalamazoo, Mich.). After fixation, the cells were washedin deionized water and incubated for two minutes in propylene glycol(absolute; Poly Scientific, Bay Shore, N.Y.). Propylene glycol wasremoved by aspiration, and samples were incubated in Oil Red 0 (PolyScientific, Bay Shore, N.Y.) for one hour. Staining solution was removedby aspiration and stained samples were then incubated in 85 percent(v/v) propylene glycol solution (Poly Scientific, Bay Shore, N.Y.) forone minute. Finally stained samples were washed with two changes ofde-ionized water. Stained samples were counter-stained with Mayer'sHematoxylin (Poly Scientific Bay Shore, N.Y.) and examined by lightmicroscopy. Images were taken at magnification of 20×.

Leptin Assay.

Adipose-derived cells and placenta-derived cells were seeded at 200,000cells/well in 6-well tissue culture-treated plates. Cells were initiallyseeded in growth medium ((DMEM:Lg; Invitrogen, Carlsbad, Calif.), 15 FBS(defined bovine serum Lot#AND18475; Hyclone, Logan, Utah), 0.001%2-mercaptoethanol (Sigma, St. Louis, Mo.), 100 Units/milliliterpenicillin, 100 microgram/milliliter streptomycin, 0.25 micrograms permilliliter amphotericin B; Invitrogen, Carlsbad, Calif.)), which waschanged to an adipogenic differentiation medium (DMEM-Hg medium (Gibco,Carlsbad, Calif.) containing 1 micromolar dexamethasone (Sigma, St.Louis, Mo.), 0.2 millimolar indomethasone (Sigma, St. Louis, Mo.), 0.01milligrams per microliter insulin (Sigma, St. Louis, Mo.), 0.5millimolar isobutylmethylxanthine (Sigma, St. Louis, Mo.), 10 percent(v/v) fetal bovine serum (Cat. #SH30070.03; Hyclone, Logan, Utah), 100Units/milliliter penicillin, 100 microgram/milliliter streptomycin(Gibco, Carlsbad Calif.)). At the end of the assay, the conditionedmedium was collected and leptin levels were measured using an ELISA kit(Quantikine, R&D Systems, Minneapolis, Minn.).

Results

Adipose Differentiation.

Morphologically, MSCs and Adipose-derived cells (Artecel; U.S. Pat. No.6,555,374) demonstrated lipid formation as early as 5 days in thisassay. Large amounts of lipid droplet formation were observed in boththese cultures by 15 days of culture. Cultures of osteoblasts alsodeposited large amounts of lipid under these conditions after 10 days inculture and extensively at 15 days. Lipid droplet formation was observedin placenta-derived and omental cell cultures after 15 days of culture.Low level lipid droplet formation was observed in the fibroblastcultures after 20 days in adipogenic-inducing conditions.

Leptin.

Leptin was not detected by ELISA in placenta-derived cell conditionedmedium.

Summary.

The potential of placenta-derived cells to differentiate into an adiposephenotype was examined. The data demonstrate that placenta-derived cellsundergo a low level of adipose differentiation when compared to culturesof mesenchymal stem cells, adipose-derived cells, or osteoblasts. Noleptin was detected in placenta-derived cells by ELISA following theadipogenic differentiation protocol used.

Example 19 Differentiation of Placenta-Derived Cells to Beta Cells

The pancreas contains endocrine cells, organized in islets ofLangerhans, which produce insulin, glucagon, somatostatin, andpancreatic polypeptide (PP). The ability of placenta-derived cells todifferentiate towards cells with an insulin-producing phenotype wastested under eight different induction protocols.

Methods & Materials

Placenta-derived cells as well as neonatal or adult Normal Human DermalFibroblasts (NHDF) were grown in Growth medium (DMEM-low glucose (Gibco,Carlsbad, Calif.), 15% (v/v) fetal bovine serum (Cat. #SH30070.03,Hyclone; Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St.Louis, Mo.), 50 Units/milliliter penicillin, 50 microgram/milliliterstreptomycin (Gibco, Carlsbad, Calif.)) in a gelatin-coated T75 flask aswell as in different beta-cell promoting differentiation conditions.Flasks were coated with 2% (w/v) gelatin solution (Sigma, St. Louis,Mo.) for 20 minutes at room temperature. Gelatin solution was aspiratedoff, and flasks were washed with PBS. Basic Fibroblast Growth Factor(bFGF), Epidermal Growth Factor (EGF), Transforming Growth Factor beta(TGFbeta) and Fibroblast Growth Factor 10 (FGF-10) were purchased fromPeproTech Inc. (Rocky Hill, N.J.). GLP-1 was purchased from Sigma (St.Louis, Mo.)

Protocol 1:

Placenta-derived cells (isolate 1; P2), adipose-derived cells (U.S. Pat.No. 6,555,374), placenta-derived cells (isolate 2; P4) (predominatelyneonatal as analyzed by karyotyping—data not shown), and adult NormalHuman Dermal Fibroblasts (NHDF) (P10). Cells were maintained undereither normal or 5% O₂ conditions. Cells were seeded at low density(5,000 cells/cm²) in gelatin-coated T75 flasks on gelatin and grown inHam's F12 medium (Clonetics, Santa Rosa, Calif.), 2% (v/v) FBS, 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin, 10nanograms/milliliter EGF, and 20 nanograms/milliliter bFGF untilconfluence. Confluent cells were trypsinized and plated at 50,000cells/cm² in 24-well Tissue Culture Polystyrene (TCPS; BD Biosciences,Bedford, Mass.) plates with or without gelatin or collagen coating.Cells were grown in Ham's F12 medium, 2% FBS, 50 Units/milliliterpenicillin, 50 microgram/milliliter streptomycin, 10nanograms/milliliter EGF, 20 nanograms/milliliter bFGF, and 15 nanoMolarGLP-1 (7-37 isoform) for up to 3 weeks.

Protocol 2:

Placenta-derived cells (isolate 3; P3) and placenta-derived cells(isolate 2; P3) (predominately neonatal as identified by karyotypinganalysis). Cells were seeded at low density (5,000 cells/cm²) in T75flasks on gelatin and grown in Ham's F12 medium, 2% FBS, 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin, 10nanograms/milliliter EGF, 20 nanograms/milliliter bFGF until confluence.Confluent cells were trypsinized and plated at 50,000 cells/cm² in24-well TCPS plates with or without gelatin coating. Cells were grown inHam's F12 medium, 2% FBS, P/S, 15 nanoMolar GLP-1 (7-37 isoform) for upto 3 weeks.

Protocol 3:

Placenta-derived cells (isolate 1; P10), adult NHDF P10, andplacenta-derived cells (isolate 2; P3). Cells were seeded at highdensity (50,000 cells/cm²) in 24-well TCPS plates and grown inDMEM:Ham's F12 (1:1) medium, B-27 supplement (Gibco, Carlsbad, Calif.),50 Units/milliliter penicillin, 50 microgram/milliliter streptomycin, 20nanograms/milliliter EGF, 40 nanograms/milliliter bFGF. Sphericalclusters were generated within about 4-6 days. Following that period,the spherical clusters were collected, centrifuged, and replated ontolaminin-coated, 24-well plates (BD Biosciences, Bedford, Mass.), andcultured up to 3 weeks in B-27-supplemented medium containing 10nanoMolar GLP-1 (7-37) with no other growth factors (i.e., no bFGF andno EGF).

Protocol 4:

Placenta-derived cells (isolate 1; P10), adult NHDF (P10),placenta-derived cells (isolate 2; P3). Cells were set up at highdensity (50,000 cells/cm²) in 24-well TCPS plates and grown inDMEM:Ham's F12 (1:1) medium, B-27 supplement, 50 Units/milliliterpenicillin, 50 microgram/milliliter streptomycin, 20nanograms/milliliter EGF, 40 nanograms/milliliter bFGF. Sphericalclusters were generated, usually in about 4-6 days. Following thatperiod, the spherical clusters were collected, centrifuged, and replatedonto laminin-coated, 24-well plates and cultured up to 3 weeks inB-27-supplemented medium containing 10 nanoMolar GLP-1 (1-37 isoform)but no other growth factors (i.e., no bFGF and no EGF).

Protocol 5:

Adult NHDF (P15) and placenta-derived cells (isolate 1; P15). Cells wereseeded at high density (50,000 cells/cm²) in 24-well TCPS gelatin-coatedplates and grown in DMEM:Ham's F12 (1:1) medium, B-27 supplement, 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin, 10nanograms/milliliter FGF-10, and/or 40 nanograms/milliliter TGFbetafor >two weeks.

Protocol 6:

Adult NHDF and placenta-derived cells (isolate 1; P15). Cells wereseeded at high density (50,000 cells/cm²) in 24-well TCPS gelatin-coatedplates and grown in EBM-2 medium, 10 nanograms/milliliter FGF-10, and/or40 nanograms/milliliter TGFbeta for >two weeks.

Protocol 7:

Placenta-derived cells (isolate 3; P3) were seeded at low density (5,000cells/cm²) in T75 flasks on gelatin and grown either in Growth medium orin Ham's F12 medium, 2% FBS, 50 Units/milliliter penicillin, 50microgram/milliliter streptomycin, 10 nanograms/milliliter EGF, 20nanograms/milliliter bFGF until confluence. Confluent cells weretrypsinized and plated at 50,000 cells/cm² in 24-well TCPS plates, withor without gelatin coating. Three types of basic media were used for upto 3 weeks:

-   -   betaI medium: Ham's F12 medium, 2% FBS, 10 millimolar        nicotinamide, 50 Units/milliliter penicillin, 50        microgram/milliliter streptomycin, 25 mM glucose    -   betaII medium: Equal parts of DMEM/Ham's F12 media, 2% FBS, 10        mM nicotinamide, 25 mM glucose    -   Endothelial Cell Basal Medium (EBM), (Clonetics, Santa Rosa,        Calif.).    -   The following growth factors were added to each of the media: 10        nanograms/milliliter EGF, 20 nanograms/milliliter bFGF, 10        nanoMolar GLP-1 (7-37 isoform).

Protocol 8:

Placenta-derived cells (isolate 2; P2) (predominately neonatal asidentified by karyotyping analysis), placenta-derived cells (isolate 2;P1), clone #22. Cells were seeded at low density (5,000 cells/cm²) inT25 TCPS flasks and grown in DMEM, 20% FBS, 50 Units/milliliterpenicillin, 50 microgram/milliliter streptomycin until confluence.

Total RNA Isolation and Quantitative RT-PCR.

RNA was extracted from placenta-derived cells and fibroblasts grown asdescribed in each protocol. Cells were lysed with 350 microliter bufferRLT containing beta-mercaptoethanol (Sigma St. Louis, Mo.) according tothe manufacturer's instructions (RNeasy Mini kit, Qiagen, Valencia,Calif.) and RNA extracted according to the manufacturer's instructions(RNeasy Mini kit, Qiagen, Valencia, Calif.) with a 2.7 Units/sampleDNase treatment (Sigma St. Louis, Mo.). RNA was eluted with 50microliter DEPC-treated water and stored at −80° C. RNA was reversetranscribed using random hexamers with the TaqMan reverse transcriptionreagents (Applied Biosystems, Foster City, Calif.) at 25° C. for 10minutes, 37° C. for 60 minutes, and 95° C. for 10 minutes. Samples werestored at −20° C.

Real-Time PCR.

PCR was performed on cDNA samples using ASSAYS-ON-DEMAND gene expressionproducts PDX-1 (Hs00426216), pro-insulin (Hs00355773), Ngn-3(Hs00360700), Glut-2 (Hs00165775), GAPDH (Applied Biosystems, FosterCity, Calif.) and TaqMan Universal PCR master mix according to themanufacturer's instructions (Applied Biosystems, Foster City, Calif.)using a 7000 sequence detection system with ABI prism 7000 SDS software(Applied Biosystems, Foster City, Calif.). Thermal cycle conditions wereinitially 50° C. for 2 minutes and 95° C. for 10 minutes followed by 40cycles of 95° C. for 15 seconds and 60° C. for 1 minute. In additionanother set of primers designed in-house for PDX-1 and Ngn-3 was tested.Table 19-1 contains primers' sequences. PCR using these primers wasperformed as described above. Pancreas total RNA (Ambion, Austin, Tex.)was used as control. PCR data was analyzed according to the ΔΔC_(T)method recommended by Applied Biosystems (User Bulletin #2 from AppliedBiosystems for ABI Prism 7700 Sequence Detection System).

TABLE 19-1 Primers Primer name Sequence PDX-1 Forward primer5′-CTGGATTGGCGTTGTTTGTG-3′ (SEQ ID NO: 11) PDX-1 Reverse primer5′-TCCCAAGGTGGAGTGCTGTAG-3′ (SEQ ID NO: 12) PDX-1-TaqMan probe5′-CTGTTGCGCACATCCCTGCCC-3′ (SEQ ID NO: 13) Ngn-3 Forward primer5′-GGCAGTCTGGCTTTCTCAGATT-3′ (SEQ ID NO: 14) Ngn-3 Reverse primer5′-CCCTCTCCCTTACCCTTAGCA-3′ (SEQ ID NO: 15) Ngn-3 TaqMan probe5′-CTGTGAAAGGACCTGTCTGTCGC-3′ (SEQ ID NO: 16)

Results

For placenta-derived cells treated according to protocols 1-8,expression of pancreas-specific marker was not detected using real-timePCR and the assay-on-demand primers, with the exception that low levelsof Ngn-3 were detected in cells from protocol 7. The same primersproduced positive results with cDNA derived from pancreatic tissue RNA.Results of real-time PCR for PDX-1 and Ngn-3 performed on cDNA samplesderived from human placenta were compared to results for adipose-derivedcells grown according to protocol 1. PCR was also performed usingin-house designed primers (Table 19-1). Results of real-time PCR usingthese primers for PDX-1 and Ngn-3 performed on cDNA samples derived fromhuman placenta were compared to results from adipose-derived cells. Dataobtained from real-time PCR was analyzed by the ΔΔC_(T) method (UserBulletin #2 from Applied Biosystems for ABI Prism 7700 SequenceDetection System) and expressed on a logarithmic scale.

Experimental conditions in Protocols 3 and 8 applied to placenta-derivedcells, but not fibroblasts, produced structures resembling the cellularassembly of pancreatic epithelial cells into islets. These structuresemerged about 3-5 days after the implementation of the protocol.Expression of pancreatic markers PDX-1, Ngn3, Glut-2 and pro-insulinwere not detected by real-time PCR.

Summary.

Limited expression of PDX-1 and Ngn-3 was observed in placenta-derivedcells treated with a variety of experimental protocols. There weredifferences in results between in-house designed and commerciallyavailable primers. For example, while protocol number 1 gave positivedata for PDX-1 and Ngn-3 using in-house designed primers,ASSAYS-ON-DEMAND primers for the same genes produced negative data. Theresults were not directly verified by immunological techniques.Notwithstanding such differences, expression of several pancreaticmarkers has been accomplished, suggesting the potential ofplacenta-derived cells to differentiate towards the pancreaticphenotypes.

Example 20 Differentiation of Placenta-Derived Cells to theCardiomyocyte Phenotype

There is a tremendous need for therapy that will slow the progression ofand/or cure heart disease, such as ischemic heart disease and congestiveheart failure. Cells that can differentiate into cardiomyocytes that canfully integrate into the patient's cardiac muscle without arrhythmiasare highly desirable. Rodent mesenchymal stem cells treated with5-azacytidine have been shown to express markers of cardiomyocytes(Fukuda et al. (2002) C. R. Biol. 325:1027-38). This has not been shownfor adult human stem cells. Additional factors have been used to improvestem cell differentiation including low oxygen (Storch (1990) Biochim.Biophys. Acta 1055:126-9), retinoic acid (Wobus et al. (1997) J. Mol.Cell Cardiol. 29:1525-39), DMSO (Xu et al. (2002) Circ. Res. 91:501-8),and chelerythrine chloride (International PCT Publication No.WO03/025149), which effects the translocation of PKC from the cytosol toplasma membrane and is an inhibitor of PKC activity. In this example,placenta-derived cells were treated with 5-azacytidine either alone orin combination with DMSO or chelerythrine chloride and markers ofcardiomyocytes measured by real-time PCR.

Methods & Materials

Cells.

Cryopreserved placenta-derived cells (P24) were grown in Growth medium(DMEM-low glucose (Gibco, Carlsbad Calif.), 15% (v/v) fetal bovine serum(Cat. #SH30070.03, Hyclone, Logan Utah), 0.001% (v/v)betamercaptoethanol (Sigma, St. Louis, Mo.), 50 Units/milliliterpenicillin, 50 microgram/milliliter streptomycin (Gibco)), in agelatin-coated flask. Cells were seeded at 5×10⁴ cells/well in 96-wellplates in Growth medium for 24 hours. The medium was changed to 0, 3, 10and 30 uM 5-azacytidine (Sigma, St. Louis, Mo.) alone or with 5microMolar chelerythrine chloride (Sigma), 1% (v/v) dimethylsulfoxide(DMSO) (Sigma), or 1 microMolar retinoic acid (Sigma) in MEM-alpha(Sigma), insulin, transferrin, and selenium (ITS; Sigma), 10% (v/v)fetal bovine serum, 50 Units/milliliter penicillin, 50microgram/milliliter streptomycin, and cells incubated at 37° C., 5%(v/v) O₂ for 48 or 72 hours. Media was then changed to MEM-alpha,insulin, transferrin, and selenium, 10% (v/v) fetal bovine serum, 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin, andcells incubated at 37° C., 5% (v/v) O₂ for 14 days.

RNA Extraction and Reverse Transcription.

Cells were lysed with 150 microliter buffer RLT containingbeta-mercaptoethanol (Sigma St. Louis, Mo.) according to themanufacturer's instructions (RNeasy 96 kit, Qiagen, Valencia, Calif.)and stored at −80° C. Cell lysates were thawed and RNA extractedaccording to the manufacturer's instructions (RNeasy 96 kit, Qiagen,Valencia, Calif.) with a 2.7 Units/sample DNase treatment (Sigma St.Louis, Mo.). RNA was eluted with 50 microliter DEPC-treated water andstored at −80° C. RNA was reverse transcribed using random hexamers withthe TaqMan reverse transcription reagents (Applied Biosystems, FosterCity, Calif.) at 25° C. for 10 minutes, 37° C. for 60 minutes and 95° C.for 10 minutes. Samples were stored at −20° C.

PCR.

PCR was performed on cDNA samples using ASSAYS-ON-DEMAND gene expressionproducts cardiac myosin (Hs00165276 ml), skeletal myosin (Hs00428600),GATA 4 (Hs00171403 ml), GAPDH (Applied Biosystems, Foster City, Calif.),and TaqMan Universal PCR master mix according to the manufacturer'sinstructions (Applied Biosystems, Foster City, Calif.) using a 7000sequence detection system with ABI prism 7000 SDS software (AppliedBiosystems, Foster City, Calif.). Thermal cycle conditions wereinitially 50° C. for 2 minutes and 95° C. for 10 minutes followed by 40cycles of 95° C. for 15 seconds and 60° C. for 1 minute. cDNA from heartand skeletal muscle (Ambion Austin Tex.) were used as a control.

Results

Control RNA from cardiac muscle showed expression of cardiac myosin andGATA 4, skeletal muscle RNA showed skeletal myosin and cardiac myosinbut no GATA 4 expression. Placenta-derived cells (P24) treated for 72 hwith factors and grown for an additional 14 days expressed GATA 4, butno skeletal myosin or cardiac myosin. Additional samples from placentathat were analyzed showed expression of GATA 4.

Summary.

Untreated placenta-derived cells constitutively express GATA 4, anuclear transcription factor in cardiomyocytes, sertoli cells, andhepatocytes.

Example 21 Treatment of Placenta-Derived Cells with Progesterone andcAMP

Placenta comprises both neonatal and maternal cells. The maternal cellsare derived from the uterine wall during the process of implantation.Endometrial cells of the uterus undergo a process called decidualizationafter conception that is driven by steroid hormones and embryonicsignals that changes the cell's morphology, phenotype and function. Themorphology of the cells changes from fibroblastic to polygonal.Expression of alpha-smooth muscle actin is reduced, and cells begin toexpress desmin, prolactin, and insulin growth factor binding protein-1(IGFBP-1) (Fazleabas and Strakova (2002) Mol. Cellular. Endo.186:143-147). In the present study the effects of progesterone and8-bromoadenosine 3′,5′-cyclicmonophosphate, a cAMP analogue, wereinvestigated. It has been previously shown that these compounds promoteendometrium decidualization in vitro (Gellersen and Brosens (2003) J.Endocrinol. 178:357-372). Fibroblasts, mesenchymal stem cells (MSC), andplacenta-derived cells were treated with progesterone and a cAMPanalogue for 3 and 6 days and stained for desmin, a marker ofdecidualization, and vimentin for mesenchymal stromal cells.

Methods & Materials

Mesenchymal stem cells (P3) (Cambrex, Walkersville, Md.),placenta-derived cells (P3) (maternal karyotype), and dermal fibroblasts(P10) (Cambrex,) were seeded onto gelatin-coated LabTek II chamberslides (Nalgene, Rochester, N.Y.) at 10,000 cells/well in Growth medium(DMEM-low glucose (Gibco Carlsbad Calif.), 15% (v/v) fetal bovine serum(Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol (Sigma, StLoius, Mo.), 50 Units/milliliter penicillin, 50 microgram/milliliterstreptomycin (Gibco)). Cells became confluent in 4 days and the mediumwas changed to either 1) control basal medium (DMEM-low glucose (Gibco),10% (v/v) fetal bovine serum charcoal/dextran-treated (Hyclone), 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin(Gibco), Fungizone (Gibco)) or 2) basal medium containing 63.5microMolar progesterone (Sigma) and 0.76 milliMolar 8-bromoadenosine3'S′-cyclicmonophosphate (Sigma). Cells were incubated for 3 or 6 dayswith media changed at 3 days. Cells were washed with PBS (Gibco) andfixed with 4% (w/v) paraformaldehyde (Sigma) for 20 minutes and storedat 4° C. in phosphate buffered saline.

Immunocytochemistry was performed to evaluate expression of vimentin(1:500, Sigma,) and desmin (1:150, Sigma). Briefly, fixed cultures werewashed with phosphate-buffered saline (PBS) and exposed to a proteinblocking solution containing PBS, 4% goat serum (Chemicon, Temecula,Calif.), and 0.3% Triton (Triton X-100, Sigma) for 30 minutes. Primaryantibody solutions were then applied to the samples containing blockingsolution plus vimentin antibody (1:500) and desmin (1:150) for a periodof 1 hour at room temperature. Next, primary antibody solutions wereremoved and samples washed with PBS prior to application of secondaryantibody solutions (1 hour at room temperature) containing blockingsolution along with goat anti-mouse IgG—Texas Red (1:250) and goatanti-rabbit IgG—Alexa 488 (1:250; Molecular Probes, Eugene, Oreg.).Samples were washed and 10 microMolar DAPI (Molecular Probes) appliedfor 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope. Representative images were captured using a digital colorvideocamera and ImagePro software (Media Cybernetics, Carlsbad, Calif.).For triple-stained samples, each image was taken using only one emissionfilter at a time. Layered montages were then prepared using AdobePhotoshop software (Adobe, San Jose, Calif.).

Results

All cells, with the exception of MSCs, in the control medium showed novimentin or desmin staining at day 3 or 6. Maternal placenta-derivedcells at 3 and 6 days showed a change in morphology when treated withprogesterone and 8-bromoadenosine 3′5′-cyclicmonophosphate.Placenta-derived cells became phase bright and had a significantlyreduced proliferation rate resulting in a lower density culture.Placenta-derived cells were the only cells to stain strongly forvimentin when treated with progesterone and 8-bromoadenosine3′5′-cyclicmonophosphate for 3 or 6 days. MSCs showed weakly positivestaining for vimentin under both conditions at 3 and 6 days.

Summary.

Placenta-derived cells and fibroblasts grown in DMEM-low glucose with10% fetal bovine serum normally express vimentin. In the presentanalysis, there was no staining for vimentin when cells were grown in10% charcoal/dextran-treated fetal calf serum for as little as 3 days.Maternal placenta-derived cells showed a change in morphology andvimentin expression with progesterone and 8-bromoadenosine3′5′-cyclicmonophosphate treatment. Expression of desmin was notdetected.

Gene chip analysis revealed that there is little or no expression of theprogesterone receptor in the cells tested. Expression of a putativesteroid receptor, progesterone membrane components 1 and 2 (Gerdes etal. (1998) Biol. Chem. 379:907-11) was detected.

Example 22 Short-Term Neural Differentiation of Placenta-Derived Cells

The ability of placenta-derived cells to differentiate into neurallineage cells was examined.

Materials & Methods

Isolation and Expansion of Placenta-Derived Cells.

Placenta-derived cells were isolated and expanded as described inExample 1.

Modified Woodbury-Black Protocol.

(A) This assay was adapted from an assay originally performed to testthe neural induction potential of bone marrow stromal cells (1).Placenta-derived cells (P3) were thawed and expanded in Growth Medium at5,000 cells/cm² until sub-confluence (75%) was reached. Cells were thentrypsinized and seeded at 6,000 cells per well of a Titretek II glassslide (VWR International, Bristol, Conn.). As controls, mesenchymal stemcells (P3; 1F2155; Cambrex, Walkersville, Md.), osteoblasts (P5; CC2538;Cambrex), omental cells (P6; (041003)), Artecel cells (U.S. Pat. No.6,555,374 B1) (P6; Donor 2) and neonatal human dermal fibroblasts (P6;CC2509; Cambrex) were also seeded under the same conditions.

All cells were initially expanded for 4 days in DMEM/F12 medium(Invitrogen, Carlsbad, Calif.) containing 15% (v/v) fetal bovine serum(FBS; Hyclone, Logan, Utah), basic fibroblast growth factor (bFGF; 20nanogram/milliliter; Peprotech, Rocky Hill, N.J.), epidermal growthfactor (EGF; 20 nanogram/milliliter; Peprotech) and 50 Units/milliliterpenicillin, 50 microgram/milliliter streptomycin (Invitrogen). After 4days, cells were rinsed in phosphate-buffered saline (PBS; Invitrogen)and were subsequently cultured in DMEM/F12 medium+20% (v/v) FBS+50Units/milliliter penicillin, 50 microgram/milliliter streptomycin for 24hours. After 24 hours, cells were rinsed with PBS. Cells were thencultured for 1 to 6 hours in an induction medium which was comprised ofDMEM/F12 (serum-free) containing 200 milliMolar butylatedhydroxyanisole, 10 nanoMolar potassium chloride, 5 milligram/milliliterinsulin, 10 nanoMolarforskolin, 4 nanoMolarvalproic acid, and 2nanoMolar hydrocortisone (all chemicals from Sigma, St. Louis, Mo.).Cells were then fixed in −20° C. 100% methanol and immunocytochemistrywas performed (see methods below) to assess human nestin proteinexpression.

(B) Placenta-derived cells (P11) and adult human dermal fibroblasts(1F1853, P11) were thawed and culture expanded in Growth Medium at 5,000cells/cm2 until sub-confluence (75%) was reached. Cells were thentrypsinized and seeded at similar density as in (A), but onto (1) 24well tissue culture-treated plates (TCP, Falcon brand, VWRInternational), (2) TCP wells+2% (w/v) gelatin adsorbed for 1 hour atroom temperature, or (3) TCP wells+20 nanogram/milliliter adsorbed mouselaminin (adsorbed for a minimum of 2 hours at 37° C.; Invitrogen).

Exactly as in (A), cells were initially expanded and media switched atthe aforementioned timeframes. One set of cultures was fixed, as before,at 5 days and six hours, this time with 4° C. 4% (w/v) paraformaldehyde(Sigma) for 10 minutes at room temperature. In the second set ofcultures, media was removed and switched to Neural Progenitor Expansionmedium (NPE) consisting of Neurobasal-A medium (Invitrogen) containingB27 (B27 supplement; Invitrogen), L-glutamine (4 milliMolar), and 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin(Invitrogen). NPE medium was further supplemented with retinoic acid(RA; 1 micromolar; Sigma). This medium was removed 4 days later andcultures were fixed with 4° C. 4% (w/v) paraformaldehyde (Sigma) for 10minutes at room temperature, and stained for nestin, GFAP, and TuJ1protein expression (see Table 22-1).

TABLE 22-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA Human Nestin 1:100Chemicon TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, MO GFAP  1:2000DakoCytomation, Carpinteria, CA Tyrosine hydroxylase (TH)  1:1000Chemicon GABA 1:400 Chemicon Desmin (mouse) 1:300 Chemicon alpha -smooth muscle actin 1:400 Sigma Human nuclear protein 1:150 Chemicon(hNuc)

Two Stage Differentiation Protocol.

Placenta-derived cells (P11), adult human dermal fibroblasts (P11;1F1853; Cambrex) were thawed and culture expanded in Growth Medium at5,000 cells/cm² until sub-confluence (75%) was reached. Cells were thentrypsinized and seeded at 2,000 cells/cm², but onto 24 well platescoated with laminin (BD Biosciences, Franklin Lakes, N.J.) in thepresence of NPE media supplemented with bFGF (20 nanogram/milliliter;Peprotech, Rocky Hill, N.J.) and EGF (20 nanogram/milliliter; Peprotech)[whole media composition further referred to as NPE+F+E]. At the sametime, adult rat neural progenitors isolated from hippocampus (P4;(062603); see Example 23)) were also plated onto 24 well laminin-coatedplates in NPE+F+E media. All cultures were maintained in such conditionsfor a period of 6 days (cells were fed once during that time) at whichtime media was switched to the differentiation conditions listed inTable 22-2 for an additional period of 7 days. Cultures were fixed withice-cold 4% (w/v) paraformaldehyde (Sigma) for 10 minutes at roomtemperature, and stained for human or rat nestin, GFAP, and TuJ1 proteinexpression.

TABLE 22-2 Summary of Conditions for Two-Stage Differentiation ProtocolA PRE- B COND. # DIFFERENTIATION 2^(nd) STAGE DIFF 1 NPE + F + E NPE +SHH (200 ng/ml) + F8 (100 ng/ml) 2 NPE + F + E NPE + SHH (200 ng/ml) +F8 (100 ng/ml) + RA (1 mM) 3 NPE + F + E NPE + RA (1 mM) 4 NPE + F + ENPE + F (20 ng/ml) + E (20 ng/ml) 5 NPE + F + E Growth Medium 6 NPE +F + E Condition 1B + rhGDF-5 (20 ng/ml) 7 NPE + F + E Condition 1B +BMP7 (20 ng/ml) 8 NPE + F + E Condition 1B + GDNF (20 ng/ml) 9 NPE + F +E Condition 2B + rhGDF-5 (20 ng/ml) 10 NPE + F + E Condition 2B + BMP7(20 ng/ml) 11 NPE + F + E Condition 2B + GDNF (20 ng/ml) 12 NPE + F + ECondition 3B + rhGDF-5 (20 ng/ml) 13 NPE + F + E Condition 3B + BMP7 (20ng/ml) 14 NPE + F + E Condition 3B + GDNF (20 ng/ml) 15 NPE + F + ENPE + rhGDF-5 (20 ng/ml) 16 NPE + F + E NPE + BMP7 (20 ng/ml) 17 NPE +F + E NPE + GDNF (20 ng/ml)

Neural Progenitor Co-Culture Protocol.

Adult rat hippocampal progenitors (062603) were plated as neurospheresor single cells (10,000 cells/well) onto laminin-coated 24 well dishes(BD Biosciences) in NPE+F (20 nanogram/milliliter)+E (20nanogram/milliliter).

Separately, placenta-derived cells (022803) P11 were thawed and cultureexpanded in NPE+F (20 nanogram/milliliter)+E (20 nanogram/milliliter) at5,000 cells/cm² for a period of 48 hours. Cells were then trypsinizedand seeded at 2,500 cells/well onto existing cultures of neuralprogenitors. At that time, existing medium was exchanged for freshmedium. Four days later, cultures were fixed with ice-cold 4% (w/v)paraformaldehyde (Sigma) for 10 minutes at room temperature, and stainedfor human nuclear protein (hNuc; Chemicon) (Table 221-1 above) toidentify PPDCs.

Immunocytochemistry.

Immunocytochemistry was performed using the antibodies listed in Table22-1. Cultures were washed with phosphate-buffered saline (PBS) andexposed to a protein blocking solution containing PBS, 4% (v/v) goatserum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma) for 30 minutes to access intracellular antigens. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. Next, primaryantibody solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing blocking solution along with goat anti-mouse IgG—Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa488 (1:250; Molecular Probes). Cultures were then washed and 10micromolar DAPI (Molecular Probes) applied for 10 minutes to visualizecell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Results

Woodbury-Black Protocol.

(A) Upon incubation in this neural induction composition, all cell typestransformed into cells with bipolar morphologies and extended processes.Other larger non-bipolar morphologies were also observed. Furthermore,the induced cell populations stained positively for nestin, a marker ofmultipotent neural stem and progenitor cells.

(B) When repeated on tissue culture plastic (TCP) dishes, nestinexpression was not observed unless laminin was pre-adsorbed to theculture surface. To further assess whether nestin-expressing cells couldthen go on to generate mature neurons, PPDCs and fibroblasts wereexposed to NPE+RA (1 microMolar), a media composition known to inducethe differentiation of neural stem and progenitor cells into such cells(2, 3, 4). Cells were stained for TuJ1, a marker for immature and matureneurons, GFAP, a marker of astrocytes, and nestin, a marker for neuralprogenitors. Under no conditions was TuJ1 expression turned on nor werecells with neuronal morphology observed, suggesting that neurons werenot generated in the short term. Furthermore, nestin and GFAP expressionwere no longer expressed by PDCs, as determined by immunocytochemistry.

Two Stage Differentiation Results.

Placenta derived cells (as well as human fibroblasts and rodent neuralprogenitors as negative and positive control cell types, respectively)were plated on laminin (neural promoting)-coated dishes and exposed to13 different growth conditions (and two control conditions) known topromote differentiation of neural progenitors into neurons andastrocytes. In addition, two conditions were added to examine theinfluence of GDF5, and BMP7 on PPDC differentiation. Generally, atwo-step differentiation approach was taken, where the cells were firstplaced in neural progenitor expansion conditions for a period of 6 daysfollowed by full differentiation conditions for 7 days. Morphologically,placenta-derived cells exhibited fundamental changes in cell morphologythroughout the time-course of this procedure. However, in no cases wereneuronal or astrocytic-shaped cells observed except for in control,neural progenitor-plated conditions. Immunocytochemistry, negative forhuman nestin, TuJ1, and GFAP confirmed these morphological observations.

Neural Progenitor and PDC Co-Culture Procedures.

Placenta-derived cells were plated onto cultures of rat neuralprogenitors seeded two days earlier in neural expansion conditions(NPE+F+E). While visual confirmation of plated placenta-derived cellsproved that these cells were plated as single cells, human-specificnuclear staining (hNuc) 4 days post-plating (6 days total length ofexposure) showed that they tended to ball up and avoid contact with theneural progenitors. Furthermore, where placental cells attached, thesecells spread out and appeared to be innervated by differentiated neuronsthat were of rat origin suggesting that the placental cells may havedifferentiated into muscle cells. This observation was based uponmorphology under phase contrast microscopy. Another observation was thattypically large cell bodies (larger than neural progenitors) possessedmorphologies resembling neural progenitors, with thin processes spanningout in multiple directions. HNuc staining (found in one half of thecell's nucleus) suggested that in some cases these human cells may havefused with rat progenitors and assumed their phenotype. Controls wellscontaining neural progenitors only had fewer total progenitors andapparent differentiated cells than did co-culture wells containingplacental cells, further indicating that placenta-derived cellsinfluenced the differentiation and behavior of neural progenitors eitherby release of chemokines and cytokines, or by contact-mediated effects.

Summary.

Multiple protocols were conducted to determine the short term potentialof placenta-derived PPDCs to differentiate into neural lineage cells.These included phase contrast imaging of morphology in combination withimmunocytochemistry for nestin, TuJ1, and GFAP, proteins associated withmultipotent neural stem and progenitor cells, immature and matureneurons, and astrocytes, respectively. Evidence was observed to suggestthat neural differentiation occurred in certain instances in theseshort-term protocols.

Several notable observations were made in co-cultures of PPDCs withneural progenitors. This approach, using human PPDCs along with axenogeneic cell type allowed for absolute determination of the origin ofeach cell in these cultures. First, some cells were observed in thesecultures where the cell cytoplasm was enlarged, with neurite-likeprocesses extending away from the cell body, yet only half of the bodylabeled with hNuc protein. Those cells may be human PPDCs that havedifferentiated into neural lineage cells or they may be PPDCs that havefused with neural progenitors of rat origin. Second, it appeared thatneural progenitors extended neurites to PPDCs in a way that indicatesthe progenitors differentiated into neurons and innervated the PPDCs.Third, cultures of neural progenitors and PPDCs had more cells of ratorigin and larger amounts of differentiation than control cultures ofneural progenitors alone, further indicating that plated PPDCs providedsoluble factors and or contact-dependent mechanisms that stimulatedneural progenitor survival, proliferation, and/or differentiation.

REFERENCES FOR EXAMPLE 22

-   (1) Woodbury, D. et al. (2000). J. Neurosci. Research. 61(4):    364-70.-   (2) Jang, Y. K. et al. (2004). J. Neurosci. Research. 75(4): 573-84.-   (3) Jones-Villeneuve, E. M. et al. (1983). Mol Cel Biol. 3(12):    2271-9.-   (4) Mayer-Proschel, M. et al. (1997). Neuron. 19(4): 773-85.

Example 23 Placenta-Derived Cellular Trophic Factors for NeuralProgenitor Support

The influence of placenta-derived cells on adult neural stem andprogenitor cell survival and differentiation through non-contactdependent (trophic) mechanisms was examined.

Materials & Methods

Adult Neural Stem and Progenitor Cell Isolation.

Fisher 344 adult rats were sacrificed by CO₂ asphyxiation followed bycervical dislocation. Whole brains were removed intact using bonerongeurs and hippocampus tissue dissected based on coronal incisionsposterior to the motor and somatosensory regions of the brain (1).Tissue was washed in Neurobasal-A medium (Invitrogen, Carlsbad, Calif.)containing B27 (B27 supplement; Invitrogen), L-glutamine (4 milliMolar;Invitrogen), and 50 Units/milliliter penicillin, 50 microgram/milliliterstreptomycin (Invitrogen), the combination of which is herein referredto as Neural Progenitor Expansion (NPE) medium. NPE medium was furthersupplemented with bFGF (20 nanogram/milliliter, Peprotech, Rocky Hill,N.J.) and EGF (20 nanogram/milliliter, Peprotech, Rocky Hill, N.J.),herein referred to as NPE+bFGF+EGF.

Following wash, the overlying meninges were removed, and the tissueminced with a scalpel. Minced tissue was collected and trypsin/EDTA(Invitrogen) added as 75% of the total volume. DNAse (100 microliter per8 milliliters total volume, Sigma, St. Louis, Mo.) was also added. Next,the tissue/media was sequentially passed through an 18 gauge needle, 20gauge needle, and finally a 25 gauge needle one time each (all needlesfrom Becton Dickinson, Franklin Lakes, N.J.). The mixture wascentrifuged for 3 minutes at 250×g. Supernatant was removed, freshNPE+bFGF+EGF was added and the pellet resuspended. The resultant cellsuspension was passed through a 40 micron cell strainer (BDBiosciences), plated on laminin-coated T-75 flasks (Becton Dickinson) orlow cluster 24-well plates (Becton Dickinson), and grown in NPE+bFGF+EGFmedia until sufficient cell numbers were obtained for the studiesoutlined.

Placenta-Derived Cell Plating.

Placenta-derived cells (P12) previously grown in Growth medium wereplated at 5,000 cells/transwell insert (sized for 24 well plate) andgrown for a period of one week in Growth medium in inserts to achieveconfluence.

Adult Neural Progenitor Plating.

Neural progenitors, grown as neurospheres or as single cells, wereseeded onto laminin-coated 24 well plates at an approximate density of2,000 cells/well in NPE+bFGF+EGF for a period of one day to promotecellular attachment. One day later, transwell inserts containingplacenta-derived cells were added according to the following scheme:

-   1) Transwell (placenta in Growth medium, 200 microliter)+neural    progenitors (NPE+bFGF+EGF, 1 milliliter)-   2) Transwell (adult human dermal fibroblasts [1F1853; Cambrex,    Walkersville, Md.] P12 in Growth medium, 200 microliter)+neural    progenitors (NPE+bFGF+EGF, 1 milliliter)-   3) Control: neural progenitors alone (NPE+bFGF+EGF, 1 milliliter)-   4) Control: neural progenitors alone (NPE only, 1 milliliter)

Immunocytochemistry.

After 7 days in co-culture, all conditions were fixed with cold 4% (w/v)paraformaldehyde (Sigma) for a period of 10 minutes at room temperature.Immunocytochemistry was performed using antibodies directed against theepitopes listed in Table 23-1. Briefly, cultures were washed withphosphate-buffered saline (PBS) and exposed to a protein blockingsolution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula,Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 30 minutes toaccess intracellular antigens. Primary antibodies, diluted in blockingsolution, were then applied to the cultures for a period of 1 hour atroom temperature. Next, primary antibodies solutions were removed andcultures washed with PBS prior to application of secondary antibodysolutions (1 hour at room temperature) containing blocking solutionalong with goat anti-mouse IgG—Texas Red (1:250; Molecular Probes,Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488 (1:250; MolecularProbes). Cultures were then washed and 10 microMolar DAPI (MolecularProbes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

TABLE 23-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA TuJ1 (BIII Tubulin)1:500 Sigma, St. Louis, MO Tyrosine hydroxylase (TH)  1:1000 ChemiconGABA 1:400 Chemicon GFAP  1:2000 DakoCytomation, Carpinteria, CA MyelinBasic Protein 1:400 Chemicon (MBP)

Quantitative Analysis of Neural Progenitor Differentiation.

Quantification of hippocampal neural progenitor differentiation wasexamined. A minimum of 1000 cells were counted per condition or if less,the total number of cells observed in that condition. The percentage ofcells positive for a given stain was assessed by dividing the number ofpositive cells by the total number of cells as determined by DAPI(nuclear) staining.

Mass Spectrometry Analysis & 2D Gel Electrophoresis.

In order to identify unique, secreted factors as a result of co-culture,conditioned media samples taken prior to culture fixation were frozendown at −80° C. overnight. Samples were then applied to ultrafiltrationspin devices (MW cutoff 30 kD). Retentate was applied to immunoaffinitychromatography (anti-Hu-albumin; IgY) (immunoaffinity did not removealbumin from the samples). Filtrate was analyzed by MALDI. The passthrough was applied to Cibachron Blue affinity chromatography. Sampleswere analyzed by SDS-PAGE and 2D gel electrophoresis.

Results

Placenta-Derived Cell Co-Culture Stimulates Adult Neural ProgenitorDifferentiation.

Following culture with placenta-derived cells, co-cultured neuralprogenitor cells derived from adult rat hippocampus exhibiteddifferentiation along all three major lineages in the central nervoussystem. This effect was clearly observed after five days in co-culture,with numerous cells elaborating complex processes and losing their phasebright features characteristic of dividing progenitor cells. Conversely,neural progenitors grown alone in the absence of bFGF and EGF appearedunhealthy and survival was limited.

After completion of the procedure, cultures were stained for markersindicative of undifferentiated stem and progenitor cells (nestin),immature and mature neurons (TuJ1), astrocytes (GFAP), and matureoligodendrocytes (MBP). Differentiation along all three lineages wasconfirmed while control conditions did not exhibit significantdifferentiation as evidenced by retention of nestin-positive stainingamongst the majority of cells. Though differentiation also appeared tobe influenced by adult human fibroblasts, such cells were not able topromote the differentiation of mature oligodendrocytes nor were theyable to generate an appreciable quantity of neurons. Though notquantified, fibroblasts did, however, appear to enhance the survival ofneural progenitors and their progeny similar to findings forplacenta-derived postpartum cells.

Identification of Unique Compounds.

Conditioned media from placental test conditions along with theappropriate controls (NPE media ±1.7% serum, media from co-culture withfibroblasts) were examined for differences. Potentially unique compoundswere identified and excised from their respective 2D gels.

Summary.

Co-culture of adult neural progenitor cells with placenta-derivedpostpartum cells results in differentiation of those cells. In view ofthe lack of contact between the PPDCs and the neural progenitors, thisresult appears to be a function of soluble factors released from thePPDCs (trophic effect).

Several other observations were made. First, there were very few cellsin the control condition where EGF and bFGF were removed. Most cellsdied and on average, there were about 100 cells or fewer per well.Second, it is to be expected that there would be very littledifferentiation in the control condition where EGF and bFGF was retainedin the medium throughout, since this is normally an expansion medium.While approximately 70% of the cells were observed to retain theirprogenitor status (nestin+), about 30% were GFAP+ (indicative ofastrocytes). This may be due to the fact that such significant expansionoccurred throughout the course of the procedure that contact betweenprogenitors induced this differentiation. Similar findings have beenreported in the literature (2).

REFERENCES FOR EXAMPLE 23

-   (1) Paxinos, G. & Watson, C. (1997). THE RAT BRAIN IN STEREOTAXIC    COORDINATES.-   (2) Song, H. et al. (2002). Nature. 417(6884): 39-44.

Example 24 Endothelial Network Formation Assay

Angiogenesis, or the formation of new vasculature, is necessary for thegrowth of new tissue. Induction of angiogenesis is an importanttherapeutic goal in many pathological conditions. The present study wasaimed at identifying potential angiogenic activity of theplacenta-derived cells in in vitro assays. The study followed awell-established method of seeding endothelial cells onto a cultureplate coated with MATRIGEL (BD Discovery Labware, Bedford, Mass.), abasement membrane extract (Nicosia and Ottinetti (1990) In Vitro CellDev. Biol. 26(2):119-28). Treating endothelial cells on MATRIGEL (BDDiscovery Labware, Bedford, Mass.) with angiogenic factors willstimulate the cells to form a network that is similar to capillaries.This is a common in vitro assay for testing stimulators and inhibitorsof blood vessel formation (Ito et al. (1996) Int. J. Cancer67(1):148-52). The protocols utilized in this example made use of aco-culture system with the placenta-derived cells seeded onto culturewell inserts. These permeable inserts allow for the passive exchange ofmedia components between the endothelial and the placenta-derivedculture media.

Material & Methods

Cell Culture.

Placenta-Derived Cells.

Human placentas were received and cells were isolated as previouslydescribed (Example 1). Cells were cultured in Growth medium (Dulbecco'sModified Essential Media (DMEM; Invitrogen, Carlsbad, Calif.), 15% (v/v)fetal bovine serum (Hyclone, Logan Utah), 100 Units/milliliterpenicillin, 100 microgram/milliliter streptomycin Invitrogen), 0.001%(v/v) 2-mercaptoethanol (Sigma, St. Louis, Mo.)) on gelatin-coatedtissue culture plastic flasks. The cultures were incubated at 37° C.with 5% CO₂. Cells used for experiments were between passages 4 and 12.

Actively growing placenta-derived cells were trypsinized, counted, andseeded onto COSTAR TRANSWELL 6.5 millimeter diameter tissue cultureinserts (Corning, Corning, N.Y.) at 15,000 cells per insert. Cells werecultured on the inserts for 48 to 72 hours in growth media in standardair with 5% CO₂ at 37° C.

Human Mesenchymal Stem Cells (hMSC).

hMSCs were purchased from Cambrex (Walkersville, Md.) and cultured inMSCGM (Cambrex). The cultures were incubated in standard air with 5% CO₂at 37° C.

Actively growing MSCs were trypsinized and counted and seeded ontoCostar® Transwell® 6.5 millimeter diameter tissue culture inserts(Corning, Corning, N.Y.) at 15,000 cells per insert. Cells were culturedon the inserts for 48 to 72 hours in growth media in standard air with5% CO₂ at 37° C.

Human Umbilical Vein Endothelial Cells (HUVEC).

HUVEC were obtained from Cambrex (Walkersville, Md.). Cells were grownin separate cultures in either EBM or EGM endothelial cell media(Cambrex). Cells were grown on standard tissue cultured plastic instandard air with 5% CO₂ at 37° C. Cells used in the assay were betweenpassages 4 and 10.

Human Coronary Artery Endothelial Cells (HCAEC).

HCAEC were purchased from Cambrex Incorporated (Walkersville, Md.).These cells were also maintained in separate cultures in either the EBMor EGM media formulations. Cells were grown on standard tissue culturedplastic in standard air with 5% CO₂ at 37° C. Cells used for experimentswere between passages 4 and 8.

Endothelial Network Formation (MATRIGEL) Assays.

Culture plates were coated with MATRIGEL (BD Discovery Labware, Bedford,Mass.) according to manufacturer's specifications. Briefly, MATRIGEL (BDDiscovery Labware, Bedford, Mass.) was thawed at 4° C. and approximately250 microliter was aliquoted and distributed evenly onto each well of achilled 24-well culture plate (Corning). The plate was then incubated at37° C. for 30 minutes to allow the material to solidify. Activelygrowing endothelial cell cultures were trypsinized and counted. Cellswere washed twice in Growth media with 2% FBS, followed bycentrifugation, resuspension, and aspiration of the supernatant. Cellswere seeded onto the coated wells 20,000 cells per well in approximately0.5 milliliter Growth medium with 2% (v/v) FBS. Cells were thenincubated for approximately 30 minutes to allow cells to settle.

Endothelial cell cultures were then treated with either 10 nanoMolarhuman bFGF (Peprotech, Rocky Hill, N.J.) or 10 nanoMolar human VEGF(Peprotech, Rocky Hill, N.J.) to serve as a positive control forendothelial cell response. Transwell inserts seeded withplacenta-derived cells were added to appropriate wells with Growthmedium with 2% FBS in the insert chamber. Cultures were incubated instandard air with 5% CO₂ at 37° C. for approximately 24 hours. The wellplate was removed from the incubator, and images of the endothelial cellcultures were collected with an Olympus inverted microscope (Olympus,Melville, N.Y.).

Results

In a co-culture system with placenta-derived cells, HUVEC form cellnetworks. HUVEC cells form limited cell networks in co-cultureexperiments with hMSC and with 10 nanoMolar bFGF. HUVEC cells withoutany treatment showed very little or no network formation. These resultssuggest that the placenta-derived cells release angiogenic factors thatstimulate the HUVEC.

In a co-culture system with placenta-derived cells, CAECs form cellnetworks.

Table 24-1 shows levels of known angiogenic factors released by PDCs inGrowth medium. Placenta-derived cells were seeded onto inserts asdescribed above. The cells were cultured at 37° C. in atmospheric oxygenfor 48 hours on the inserts and then switched to a 2% FBS medium andreturned at 37° C. for 24 hours. Media was removed, immediately frozenand stored at −80° C., and analyzed by the SEARCHLIGHT multiplex ELISAassay (Pierce Chemical Company, Rockford, Ill.). Results shown are theaverages of duplicate measurements. The results show that theplacenta-derived cells do not release detectable levels ofplatelet-derived growth factor-bb (PDGF-bb) or heparin-binding epidermalgrowth factor (HBEGF). The cells do release measurable quantities oftissue inhibitor of metalloprotease-1 (TIMP-1), angiopoietin 2 (ANG2),thrombopoietin (TPO), keratinocyte growth factor (KGF), hepatocytegrowth factor (HGF), fibroblast growth factor (FGF), and vascularendothelial growth factor (VEGF).

TABLE 24-1 Potential angiogenic factors released from placenta-derivedcells. Cells were cultured in 24 hours in media with 2% FBS inatmospheric oxygen. Media was removed and assayed by the SEARCHLIGHTmultiplex ELISA assay (Pierce). Results are the means of a duplicateanalysis. Values are concentrations in the media reported in picogramsper milliliter of culture media. TIMP1 ANG2 PDGF-BB TPO KGF HGF FGF VEGFHB-EGF (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml)(pg/ml) Plac (P4) 91655.3 175.5 <2.0 275.5 3.0 58.3 7.5 644.6 <1.2 Plac(P11) 1592832.4 28.1 <2.0 1273.1 193.3 5960.3 34.8 12361.1 1.7 Mediaalone <9.8 25.1 <2.0 <6.4 <2.0 <3.2 <5.4 <4.0 <1.2

Table 24-2 shows levels of known angiogenic factors released by PDCs.PDCs were seeded onto inserts as described above. The cells werecultured in Growth medium at 5% oxygen for 48 hours on the inserts andthen switched to a 2% FBS medium and returned to 5% O₂ incubation for 24hours. Media was removed, immediately frozen, and stored at −80° C., andanalyzed by the SEARCHLIGHT multiplex ELISA assay (Pierce ChemicalCompany, Rockford, Ill.). Results shown are the averages of duplicatemeasurements. The results show that the placenta-derived cells do notrelease detectable levels of platelet-derived growth factor-bb(PDGF-BB), or heparin-binding epidermal growth factor (HBEGF). The cellsdo release measurable quantities of tissue inhibitor ofmetallinoprotease-1 (TIMP-1), angiopoietin 2 (ANG2), thrombopoietin(TPO), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF),fibroblast growth factor (FGF) and vascular endothelial growth factor(VEGF).

TABLE 24-2 Potential angiogenic factors released from placenta-derivedcells. Cells were cultured in 24 hours in media with 2% FBS in 5%oxygen. Media was removed and assayed by the SEARCHLIGHT multiplex ELISAassay (Pierce). Results are the means of a duplicate analysis. Valuesare concentrations in the media reported in picograms per milliter ofculture media. TIMP1 ANG2 PDGF-BB TPO KGF HGF FGF VEGF HB-EGF (pg/ml)(pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) Plac(P4) 72972.5 253.6 <2.0 743.1 2.5 30.2 15.1 1495.1 <1.2 Plac (P11)458023.1 55.1 <2.0 2562.2 114.2 2138.0 295.1 7521.3 1.8 Media alone <9.825.1 <2.0 <6.4 <2.0 <3.2 <5.4 <4.0 <1.2

Summary.

The results show that placenta-derived cells can stimulate both humanumbilical vein and coronary artery endothelial cells to form networks inan in vitro MATRIGEL (BD Discovery Labware, Bedford, Mass.) assay. Thiseffect is similar to that seen with known angiogenic factors in thisassay system. These results suggest that PDCs are useful for stimulatingangiogenesis in vivo.

Example 25 Transplantation of Placenta-Derived Cells Under the KidneyCapsule

Transplantation of pancreatic islets to the kidney capsule is routinelyperformed to evaluate transplantation methodologies for the treatment ofdiabetes (Refaie et al. (1998) Trans. Proc. 30:400-403). In addition topancreatic islets, other cells may be differentiated intoinsulin-secreting cells capable of blood glucose homeostasis. Thepurpose of this study was to determine whether cells derived from humanplacenta could survive when implanted under the kidney capsule inimmune-deficient mice. In addition, placenta-derived cells were mixedwith GM-CSF mobilized CD34+ cells to determine whether these cells couldpromote vascularization and survival of the placenta-derived cells.

Methods & Materials

Cell Culture.

Cryopreserved placenta-derived cells (isolate 1, P10) were removed fromliquid nitrogen storage and grown in Growth medium (DMEM-low glucose(Gibco Carlsbad Calif.), 15% (v/v) fetal bovine serum (Hyclone, Logan,Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St Louis, Mo.), 50Units/milliliter penicillin, 50 microgram/milliliter streptomycin(Gibco)) on gelatin (Sigma)-coated T225 (Corning, Corning, N.Y.) flasksuntil confluent.

Cells from two flasks were washed with Phosphate buffered saline (PBS)and a single cell suspension was obtained by using Trypsin/EDTA (Gibco).Cryopreserved GM-CSF mobilized CD34+ cells were purchased from Cambrex,Walkersville, Md. (lot 1F0174 donor 7956). CD34+ cells were thawed andwashed in DMEM medium.

The cell suspension was washed twice in DMEM. Cell number and viabilitywas estimated after trypan blue (Sigma) staining using a hemocytometer.Aliquots of the cell suspension containing ˜300,000 viable cells werecentrifuged at 150×g, and the cells were resuspended in approximately 6microliter of DMEM and drawn into a 20 microliter pipette tip connectedto a 1 milliliter syringe. The tip of the pipette tip containing thecells was clamped using a small Ligaclip (Ethicon Endosurgery,Cincinnati Ohio).

Animal Preparation.

Mice (Mus Musculus)/Fox Chase SCID/Male (Harlan Sprague Dawley, Inc.,Indianapolis, Ind.), 8 Weeks of Age.

All handling of the SCID mice took place under a hood. The mice wereindividually weighed and anesthetized with an intraperitoneal injectionof a mixture of 60 milligrams/kilogram KETASET (ketamine hydrochloride,Aveco Co., Inc., Fort Dodge, Iowa) and 10 milligrams/kilogram ROMPUN(xylazine, Mobay Corp., Shawnee, Kans.) and saline. After induction ofanesthesia, the entire back of the animal from the dorsal cervical areato the dorsal lumbosacral area was clipped free of hair using electricanimal clippers. The area was scrubbed with chlorhexidine diacetate,rinsed with alcohol, dried, and painted with an aqueous iodophorsolution of 1% available iodine. Ophthalmic ointment was applied to theeyes to prevent drying of the tissue during the anesthetic period. Theanesthetized and surgically prepared animal was placed in the desiredrecumbent position. A transverse incision was made on the left abdominalside approximately 2 cm caudal to the rib cage of animal. The kidney wasexposed and the capsule pierced with a 26-gauge needle. A capsule lance(modified glass pipette tip) was used to create a space beneath thekidney capsule into which the cells were introduced. The cells wereinjected via a syringe with a micropipette tip attached. The pocket wasclosed by passing an ophthalmic cautery pen (Aaron medical Industries,St. Petersburg, Fla.) over the opening (not touching the kidney). Thekidney was placed back in the correct anatomical position and the musclelayer sutured closed. The skin was closed with wound clips.

The experimental design comprised one transplantation of cells per mouse(Table 25-1); four treatments with n-value of 4 per treatment; and threetime-points (1, 14, and 30 days).

Mice were euthanized at their designated intervals by carbon dioxideinhalation. The kidney implantation sites were excised and frozen forhistology.

Immunohistochemistry.

Frozen kidney implantation sites were embedded on edge in O.C.T.Compound (Sakura, Torrance, Calif.). The kidney tissue was trimmed bycryosectioning to yield a five-micron section of the implantation siteand adjacent tissue. Yielded sections were fixed in freshly prepared 4%paraformaldehyde (EM Sciences Gibbstown, N.J.) in phosphate bufferedsaline (Gibco) for 15 minutes. Sections were washed in PBS and incubatedin 3% goat serum in PBS blocking solution for one hour. Blockingsolution was removed by gentle aspiration. Sections were incubated inanti-human nuclei antibody (Chemicon International, Temecula, Calif.)diluted 1:100 in blocking solution for one hour. Sections were washedwith PBS and incubated in fluorescent labeled goat anti-mouse IgGantibody (Molecular Probes Eugene, Oreg.) diluted 1:200 in blockingsolution for 30 minutes in absence of light. Sections were washed in PBSand incubated in 10 microMolar DAPI (Molecular Probes Eugene, Oreg.) forfive minutes. Sections were washed in PBS and examined by fluorescentmicroscopy.

Tri-Chrome Staining.

Frozen kidney implantation sites were embedded on edge in O.C.T.Compound (Sakura Torrance, Calif.). The kidney tissue was trimmed bycryosectioning to yield a five-micron section of the implantation siteand adjacent tissue. Yielded sections were fixed in 10% neutral bufferedformalin (Richard-Allan Scientific Kalamazoo, Mich.) for 15 minutes.Sections were stained tri-chrome (Poly Scientific Bay Shore, N.Y.) usingmanufacturer's methods.

TABLE 25-1 SCID Mouse Kidney Capsule Cell Transplantation Scheme Animal(#) Post-Transplantation Days Kidney Capsule (left) 1 1 1 2 1 1 3 1 1 41 1 5 14 1 6 14 1 7 14 1 8 14 1 9 30 1 10 30 1 11 30 1 12 30 1 13 1 2 141 2 15 1 2 16 1 2 17 14 2 18 14 2 19 14 2 20 14 2 21 30 2 22 30 2 23 302 24 30 2

Treatments:

1. 3×10³ cells from placenta

2. 3×10³ cells from placenta+3×10³ CD34+ cells

Added animal #25-27 as control (No cells)

Results

The viability of the placenta-derived cells was ˜75% and the CD34+ cellswas 95%. Initial attempts to transplant 1×10⁶ viable cells wereunsuccessful due to inadequate size of the kidney capsule to accommodatethe cells. Cells were transplanted within 3 hours of trypsinization. Thelocalization of placenta-derived cells under the kidney capsule wasobserved microscopically. There were no apparent differences in thenumber and distribution of placenta-derived cells with or without CD34+cells at each time point. There was an apparent decrease in cell numbersover time.

Staining of cells under the kidney capsule showed the retention oftransplanted cells. Human cells were detected using the human nuclearantigen. All cells (human and mouse) were detected using DAPI.

Summary.

Transplantation of cells into the renal capsule was successful.Undifferentiated placenta-derived cells (3×10³) pre-treated with growthfactors with or without 3×10³ GM-CSF mobilized CD34+ cells weretransplanted beneath the capsule of the kidney. Animals were sacrificedat 1, 14, and 30 days following cell transplantation. Cells survived at1, 14, and 30 days with a reduction in apparent cell numbers at 30 days.The presence of GM-CSF mobilized CD34+ cells did not effect the survivalof placenta-derived cells. This study demonstrates that placenta-derivedcells can be transplanted to the kidney capsule.

Example 26 Transplantation of Placenta-Derived Cells

Cells derived from the postpartum placenta are useful for regenerativetherapies. The tissue produced by placenta-derived cells transplantedinto SCID mice with a biodegradable material was evaluated. Thematerials evaluated were VICRYL nonwoven, 35/65 PCL/PGA foam, and RAD 16self-assembling peptide hydrogel.

Methods & Materials

Cell Culture.

Placenta-derived cells were grown in Growth medium (DMEM-low glucose(Gibco, Carlsbad Calif.), 15% (v/v) fetal bovine serum (Cat.#SH30070.03; Hyclone, Logan, Utah), 0.001% (v/v) betamercaptoethanol(Sigma, St. Louis, Mo.), 50 Units/milliliter penicillin, 50microgram/milliliter streptomycin (Gibco)) in a gelatin-coated flasks.

Matrix Preparation.

A nonwoven scaffold was prepared using a traditional needle punchingtechnique as described below. Fibers, comprised of a syntheticabsorbable copolymer of glycolic and lactic acids (PGA/PLA), sold underthe tradename VICRYL were obtained from Ethicon, Inc. (Somerville,N.J.). The fibers were filaments of approximately 20 microns indiameter. The fibers were then cut and crimped into uniform 2-inchlengths to form 2-inch staple fiber. A dry lay needle-punched nonwovenmatrix was then prepared utilizing the VICRYL staple fibers. The staplefibers were opened and carded on standard nonwoven machinery. Theresulting mat was in the form of webbed staple fibers. The webbed staplefibers were needle-punched to form the dry lay needle-punched nonwovenscaffold. The nonwoven scaffold was rinsed in water followed by anotherincubation in ethanol to remove any residual chemicals or processingaids used during the manufacturing process.

Foams, composed of 35/65 poly(epsilon-caprolactone)/poly(glycolic acid)(35/65 PCL/PGA) copolymer, were formed by the process of lyophilized, asdiscussed in U.S. Pat. No. 6,355,699.

Sample Preparation.

One million viable cells were seeded in 15 microliter Growth medium onto5 millimeter diameter, 2.25 millimeter thick nonwoven scaffolds (64.33milligram/cubic centimeters) or 5 millimeter diameter 35/65 PCL/PGA foamdisks. Cells were allowed to attach for two hours before adding moreGrowth medium to cover the scaffolds. Cells were grown on scaffoldsovernight. Scaffolds without cells were also incubated in medium.

RAD16 self-assembling peptides (3D Matrix, Cambridge, Mass. under amaterial transfer agreement) was obtained as a sterile 1% (w/v) solutionin water, which was mixed 1:1 with 1×10⁶ cells in 10% (w/v) sucrose(Sigma, St Louis, Mo.), 10 millimolar HEPES in Dulbecco's modifiedmedium (DMEM; Gibco) immediately before use. The final concentration ofcells in RAD16 hydrogel was 1×10⁶ cells/100 microliter.

Test Material (N=4/Rx)

1. VICRYL nonwoven+1×10⁶ placenta-derived cells

2. 35/65 PCL/PGA foam+1×10⁶ placenta-derived cells

3. RAD 16 self-assembling peptide+1×10⁶ placenta-derived cells

4. 35/65 PCL/PGA foam

5. VICRYL nonwoven

Animal Preparation.

The animals utilized in this study were handled and maintained inaccordance with the current requirements of the Animal Welfare Act.Compliance with the above Public Laws were accomplished by adhering tothe Animal Welfare regulations (9 CFR) and conforming to the currentstandards promulgated in the Guide for the Care and Use of LaboratoryAnimals, 7th edition.

Mice (Mus Musculus)/Fox Chase SCID/Male (Harlan Sprague Dawley, Inc.,Indianapolis, Ind.), 5 Weeks of Age.

All handling of the SCID mice took place under a hood. The mice wereindividually weighed and anesthetized with an intraperitoneal injectionof a mixture of 60 milligram/kilogram KETASET (ketamine hydrochloride,Aveco Co., Inc., Fort Dodge, Iowa) and 10 milligram/kilogram ROMPUN(xylazine, Mobay Corp., Shawnee, Kans.) and saline. After induction ofanesthesia, the entire back of the animal from the dorsal cervical areato the dorsal lumbosacral area was clipped free of hair using electricanimal clippers. The area was then scrubbed with chlorhexidinediacetate, rinsed with alcohol, dried, and painted with an aqueousiodophor solution of 1% available iodine. Ophthalmic ointment wasapplied to the eyes to prevent drying of the tissue during theanesthetic period.

Subcutaneous Implantation Technique.

Four skin incisions, each approximately 1.0 cm in length, were made onthe dorsum of the mice. Two cranial sites were located transversely overthe dorsal lateral thoracic region, about 5-millimeter caudal to thepalpated inferior edge of the scapula, with one to the left and one tothe right of the vertebral column. Another two were placed transverselyover the gluteal muscle area at the caudal sacro-lumbar level, about5-mm caudal to the palpated iliac crest, with one on either side of themidline. Implants were randomly placed in these sites. The skin wasseparated from the underlying connective tissue to make a small pocketand the implant placed (or injected for RAD16) about 1-cm caudal to theincision. The appropriate test material was implanted into thesubcutaneous space. The skin incision was closed with metal clips.

Animal Housing.

Mice were individually housed in microisolator cages throughout thecourse of the study within a temperature range of 64° F.-79° F. andrelative humidity of 30% to 70% and were maintained on an approximate 12hour light/12 hour dark cycle. The temperature and relative humiditywere maintained within the stated ranges to the greatest extentpossible. Diet consisted of Irradiated Pico Mouse Chow 5058 (Purina Co.)and water fed ad libitum.

Mice were euthanized at their designated intervals by carbon dioxideinhalation. The subcutaneous implantation sites with their overlyingskin were excised and frozen for histology.

Histology.

Excised skin with implant was fixed with 10% neutral buffered formalin(Richard-Allan Kalamazoo, Mich.). Samples with overlying and adjacenttissue were centrally bisected, paraffin-processed, and embedded on cutsurface using routine methods. Five-micron tissue sections were obtainedby microtome and stained with hematoxylin and eosin (Poly Scientific BayShore, N.Y.) using routine methods.

Results

There was minimal ingrowth of tissue into foams implanted subcutaneouslyin SCID mice after 30 day. In contrast there was extensive tissue fillin foams implanted with placenta-derived cells.

There was some tissue in growth in VICRYL nonwoven scaffolds. Nonwovenscaffolds seeded with placenta-derived cells showed increased matrixdeposition and mature blood vessels.

It was not possible to identify the point of injection of RAD16 andcells.

Summary.

The purpose of this study was to determine the type of tissue formed bycells derived from human placenta in scaffolds in immune deficient mice.Synthetic absorbable nonwoven/foam discs (5.0 millimeter diameter×1.0millimeter thick) or self-assembling peptide hydrogel were seeded withcells derived from human placenta and implanted subcutaneouslybilaterally in the dorsal spine region of SCID mice. It has beendemonstrated that placenta-derived cells can dramatically increase goodquality tissue formation in biodegradable scaffolds.

Example 27 Assessment of Placenta-Derived Cells for CardiovascularTherapy in a Rodent Coronary Ligation Model

The efficacy of intracardiac human placenta-derived cell treatment whenadministered 15 minutes post-coronary artery occlusion was evaluated ina rodent model of myocardial ischemia/infarction.

Methods & Materials

The Charles River Worcester, Mass. test facility is accredited by theAssociation for the Assessment and Accreditation of Laboratory AnimalCare, International (AAALAC) and registered with the United StatesDepartment of Agriculture to conduct research in laboratory animals. Allthe conditions of testing will conform to the Animal Welfare Act (9 CFR)and its amendments. The protocol was reviewed and approved by theInstitutional Animal Care and Use Committee (IACUC) at the Test Facilityfor compliance with regulations prior to study initiation.

The animals having characteristics identified in Table 27-1 wereindividually housed in micro-isolator cages on autoclaved bedding. Thecages conform to standards set forth in The Guide for the Care and Useof Laboratory Animals.

TABLE 27-1 Animal characteristics Species: Rattus norvegicus Strain: RnuSource: Charles River Laboratories Age at Dosing: 6-8 weeks Weight atDosing: ~200-250 grams Number of Males (including spares): 40 + 10

Purina Certified Diet (irradiated) was provided to the animals adlibitum. This diet was routinely analyzed by the manufacturer fornutritional components and environmental contaminants. Results of themanufacturer's analyses are on file at the Test Facility. AutoclavedFiltered tap water was provided ad libitum. Samples of the filteredwater were analyzed for total dissolved solids, hardness, specifiedmicrobiological content, and selected environmental contaminants.Results of these analyses are on file at the Test Facility.

Environmental controls were set to maintain temperatures of 18 to 26° C.(64 to 79° F.) with a relative humidity of 30% to 70%. A 12:12 hourlight:dark cycle was maintained. Ten or greater air changes per hourwere maintained in the animal rooms. Upon receipt and prior to use onthe study, the animals were held for a minimum of four days forconditioning according to the Test Facility Vendor Management Program asdescribed in the Test Facility Standard Operating Procedure, Receipt,Conditioning, and Quarantine of Laboratory Animals.

Each animal was identified by a unique number and this number wasindicated by an ear punch. Animals were randomly assigned to groups by aweight-ordered distribution such that individual body weights did notexceed ±20% of mean weight.

The animals were anesthetized with sodium pentobarbital (40milligram/kilogram) and buprenorphine (0.05 milligram/kilogram) as asingle cocktail given intramuscularly (IM). Following the establishmentof anesthesia, animals were intubated using an 18-16 gauge, 2-inchlength angiocath, or appropriate sized angiocath, and maintained on roomair respiration (supplemented with oxygen) and a positive pressureventilator throughout the surgical procedure. Additional anesthesia wasgiven incrementally as needed. Preoperative antibiotic therapy was alsoadministered, Benzathine/Procaine penicillin G, 40,000 Units/kilogram,IM. Additional antibiotic therapy was administered every 48 hours.

Electrode pads were placed around the appropriate paws of the animals toreceive a useable electrocardiogram (ECG) signal. Animals werepositioned on a heating pad to help maintain body temperature throughoutthe procedure. A rectal temperature probe was inserted into the animalto monitor body temperature. Ophthalmic ointment was administered toeach eye. The surgical sites (thoracic area) were prepared for asepticsurgery by removing any excess fur, and gently wiping the area withsponges that have been soaked in 70% isopropyl alcohol, which wasallowed to dry. Medi Sepps™ or similar solution was then applied to thearea and also allowed to dry. The area was appropriately draped forstrict aseptic surgery.

A surgical incision was made on the skin over the fourth intercostalspace. Blunt dissection through the muscle layers was used to access thethoracic cavity. A retractor was carefully inserted into the fourthintercostal space and opened to allow access to the interior cavity. Thepericardium was carefully opened via gentle teasing with cotton swabsdampened in sterile saline solution. A damp cotton swab was used togently push the apex of the heart into the opening where a length of 6-0silk suture was attached into the myocardium for manipulation of theheart. After a pause to allow the heart to recover, the suture placed inthe apex was used to ease the heart out of the chest cavity and to placesufficient tension on the heart to allow access to the upper heart andthe left anterior descending coronary artery (LAD). Another length of6-0 silk suture was placed into the myocardium so as to surround theLAD. The pressure on the apical suture was released and the heartallowed to return to the interior of the chest cavity.

Once the heart rate and ECG returned to baseline values, the ligaturesaround the LAD were tied off to occlude the LAD. This was a permanentocclusion with the suture tied off and the ends trimmed. Once theligature was tied, the surgeon looked for the following indications ofsuccessful occlusion: change in color of the area of the heart directlybelow the ligature to a white/grayish white as a result of thetermination of blood flow to the area and a significant change in theECG corresponding to occlusion of the LAD. Arrhythmias may havedeveloped within the first 10 minutes of the occlusion. The rat wasmonitored closely during this time period in the event thatresuscitation was necessary. In the event of severe arrhythmia andfailure of the rat to convert to normal sinus rhythm without assistance,aid was rendered via cardiac massage. Approximately 15 minutes followingthe initiation of the LAD occlusion, the area of left ventricle madeischemic was treated with either vehicle or test article by directinjection into the ischemic myocardium. Treatment consisted of three toten intramyocardial injections (100 microliter/injection) into theischemic zone of myocardium.

Human cells were grown in Growth medium (DMEM-low glucose (Gibco,Carlsbad Calif.), 15% (v/v) fetal bovine serum (Cat. #SH30070.03,Hyclone, Logan Utah), 0.001% (v/v) betamercaptoethanol (Sigma, St.Louis, Mo.), 50 Units/milliliter penicillin, 50 microgram/milliliterstreptomycin (Gibco, Carlsbad Calif.), in a gelatin-coated T300 flasks.Cells were washed with phosphate buffered saline (PBS, Gibco, CarlsbadCalif.) and trypsinized using Trypsin/EDTA (Gibco, Carlsbad Calif.). Thetrypsinization was stopped by adding Growth medium. The cells werecentrifuged at 150×g, supernatant removed, and the cell pellet wasresuspended in approximately 1 milliliter Growth medium per millioncells. An aliquot of cells was removed and added to trypan blue (Sigma,St. Louis, Mo.). The viable cell number was estimated using ahemocytometer. The cell suspension was centrifuged and resuspended in 1milliliter Growth containing 10% (v/v) DMSO (Hybrimax, Sigma, St. Louis,Mo.) per 5 million cells and transferred into Cryovials (Nalgene). Thecells were cooled at approximately 1° C./minute overnight in a −80° C.freezer using a “Mr Frosty” freezing container (Nalgene, Rochester,N.Y.). Vials of cells were transferred into liquid nitrogen. Vials wereshipped from CBAT, Somerville, N.J. to Charles River, Worcester, Mass.on dry ice and stored at −80° C. Approximately 1-2 hours beforeinjection of cells into the animal, a vial of cells was thawed rapidlyin a 37° C. water bath. Under aseptic conditions in a BSL2 biosafetycabinet, cells were added to 40 milliliters PBS with magnesium andcalcium (Sigma St. Louis, Mo.) and centrifuged at 150×g for 5 minutesbefore resuspending the cell pellet in 10 milliliters PBS. The cellnumber and viability was estimated as described above. The cells werecentrifuged at 150×g for 5 minutes and resuspended in PBS at a finalconcentration of 10⁶ viable cells/100 microliter. The cell suspensionwas loaded into 1 milliliter syringes with a 30 G needle and kept onice. Viability was assessed again up to 5 hours on ice.

Following the administration of treatment (Table 27-2) and stabilizationof the heart, the surgeon began closing the surgical incision. Theretractor was removed. The lungs were over-inflated for 3-4 breaths andvisually inspected as much as possible to ensure that they were fullyre-inflated. This created a negative pressure necessary to preventpneumothorax post-recovery. To evacuate fluid and excess air from thethoracic cavity after closing the cavity, an intravenous catheter (i.e.,20 gauge, 2 millimeter in length) was placed through the skin and musclelayers so that the tip remains in the thoracic cavity. Care was taken sothat the tip did not pierce the lung or heart. The separated ribs andassociated muscle was sutured together with appropriate suture. Theupper layers of muscle was sutured using a simple continuous pattern.The skin was closed with 4-0 silk using a horizontal mattress pattern. A10 milliliter syringe was attached to the intravenous catheter that hadbeen previously placed in the thoracic cavity and the plunger slowlypulled back to withdraw fluids and air from the cavity. At the sametime, the catheter was slowly withdrawn from the entry site, therebyallowing the surrounding muscle mass and skin to seal the puncture. Thesurgical drape was removed and fluids (i.e., lactated Ringers solution,25 milliliter/kilogram subcutaneously [SC] or intraperitoneally [IP])were given.

TABLE 27-2 Treatment regimens Time of Gr. No. of Dosage Level Dose Conc.Route/Dose Treatment Necropsy No. Males Test Article (cells/animal)(cells/mL) Regimen Administration Day 1 8 Vehicle 0 0 Direct 15 minutesDay 28 2 8 Placenta #4 1 million 10 million injection(s) into aftercoronary (±1 Day) (P10) (A) the ischemic artery ligation 3 8 Placenta #3region of the left (P10) (C) ventricle of the 4 8 Human fibroblastsheart, consisting 1F1853 (P10) (D) of 3 to 10 intramyocardial injectionsof 100 microliters total. Gr. = Group; No. = Number; Conc. =Concentration

Immediately after each rat had undergone treatment with test article andthe incision sutured, the animal underwent an echocardiography (ECG)examination. Anesthesia was maintained throughout the completion of theecho examination. Upon the completion of the echo examination,ventilation was discontinued, and the rat was returned to the recoveryarea to recover in a heated, oxygenated recovery cage.

A second echo examination of each surviving animal was completed at theend of the study (approximately 28 days post-treatment), prior totermination. During the second examination, the animals wereanesthetized as described previously.

For each echo examination, the left thoracic area was shaved, andwarmed, ultrasonic gel was applied to the skin to enhance contact withthe transducer. Electrode pads were placed around the appropriateextremities to receive an ECG signal. Echocardiographic images includedshort axis and long axis views to allow for the determination ofventricular cavity dimensions, contractility, blood flow throughvasculature, and wall thickness. These images were saved on optical diskfor further analysis. After examination, the gel medium was removed fromthe skin with gauze or paper towel. The rat was removed from theventilator and placed in a warmed recovery cage until mobile.

At the conclusion of the surgical procedures, respiratory ventilationwas turned off. The animals were observed for pedal reflex. The rectalprobe and ECG electrodes subsequently were removed, and the animal wasextubated and placed in a warmed oxygenated recovery cage. Aftercomplete recovery from anesthesia, the animals were given buprenorphine(0.05 milligram/kilogram, SC). Observations were made regularly untilthe animals showed full mobility and an interest in food and water. Theanimals then were placed in a clean housing cage and returned to theanimal housing room. Animals were monitored for surgical incisionintegrity twice daily post-surgery.

Analgesics (i.e., Buprenorphine, 0.05 milligram/kilogram SC) were giventwice daily for 4 days post-operatively and thereafter as needed. Visualindications of post-operative pain include lack of normal body posturesand movement (e.g., animal remains in hunched position), antipathy, lackof eating/drinking, lack of grooming, etc.

Body weight was recorded for each animal prior to initial treatment,weekly thereafter, and on the day of necropsy. Animals found dead wereweighed and necropsied.

In order for the heart to be harvested, each rat was anesthetized as wasdone for surgery. The jugular vein was cannulated. The heart wasarrested in diastole with potassium chloride infused via the jugularcannula. The heart was then removed from the thoracic cavity. A limitednecropsy was then performed on the heart after which the heart wasplaced in 10% neutral buffered formalin. The remainder of each carcasswas then discarded with no further evaluation.

Hearts of all animals that were found dead or euthanized moribund wereplaced in 4% paraformaldehyde until evaluated. The remainder of eachcarcass was then discarded with no further evaluation.

Histology and Image Analysis.

Fixed tissues sectioned with a stainless steel coronal heart matrix(Harvard Apparatus Holliston, Mass.) yielded four two-millimeter thickserial tissue sections. Sections were processed and serially embedded inparaffin using routine methods. Five-micron sections were obtained bymicrotome and stained Masson's Tri-chrome for Connective Tissue (PolyScientific Bay Shore, N.Y.) using manufacturer's methods. Electronicphotomicrographs were captured and analyzed using image analysis methodsdeveloped by Phase 3 Imaging System (Glen Mills, Pa.). Photomicrographsof the tri-chrome stained sections were color-metrically analyzedelectronically to determine the overall area of the ventricle and freewall and the area of the differential staining.

Results

There was no loss in the initial viability of cells over 5 hours in thevehicle when kept on ice. Cells were injected into the infarct with oneto three needle entry points and multiple changes in direction of needleorientation.

Echocardiography measurements were taken from the infarct-treated rats.Fractional shortening of the vehicle-treated animals had a significantdecrease from 47.7%±8.3% at Day 0 to 23.5%±30.2% at Day 28 (p<0.05). Theanimals that were treated with placenta-derived cells showed small,non-significant differences between the fractional shortening betweenDay 0 and 28. There was no significant difference between the fractionalshortening between the groups at Day 0. Each group had eight animals atthe start but some did not survive the experiment. Thefibroblast-treated animals experienced greater mortality than thosetreated with PDCs.

Hearts collected at the study termination were subjected to histologicalanalysis. The hearts were arrested in diastole and fixed. The resultswere calculated from an algorithm to estimate the percentage of totalheart area that comprises the infarct. The infarct size in thevehicle-treated animals was 22.9%±6.7% of heart area, while the infarctsize in hearts treated with two different isolates of placenta-derivedcells was 13.9%±3.7% and 12.9%±3.4%, respectively, and with fibroblastswas 19.3%±8.0%. The difference of infarct size of cell-treated animalsrelative to vehicle-treated animals was not statistically significant,as determined by t-test/ANOVA.

Summary.

The results of the present study suggest that the placenta-derived cellshave some benefit in reducing the damage of a surgically inducedmyocardial infarction in rats. The vehicle-treated animals showed asignificant reduction in cardiac function at day 28 as compared to day0, as measured by fractional shortening, while the placenta-derivedcell-treated animals showed minimal change over the 28-day study. Thefibroblast-treated animals showed minimal change but only two animalssurvived the study. Evaluation of infarct size suggested that there maybe some modest, but not statistically significant, reduction in theinfarct size in the placenta-derived cell-treated animals as compared tothe vehicle controls at Day 28. Taken together, these data supportefficacy of the placenta-derived cells in reducing damage from amyocardial infarction.

Example 28 Use of Placenta-Derived Cells in the Treatment of RetinitisPigmentosa

Currently no real treatment exists for blinding disorders that stem fromthe degeneration of cells in the retina. Loss of photoreceptors as aresult of apoptosis or secondary degeneration lead to progressivedeterioration of vision, and ultimately to blindness. Diseases in whichthis occurs include age-related macular degeneration (AMD) and retinitispigmentosa (RP). RP is most commonly associated with a single genemutation, which contributes to photoreceptor cell death.

The retinal photoreceptors and adjacent retinal pigment epithelium forma functional unit. The Royal College of Surgeons (RCS) rat presents witha tyrosine receptor kinase (Merkt) defect affecting outer segmentphagocytosis, leading to photoreceptor cell death (D'Cruz et al. (2000)Hum. Mol. Genet. 9(4):645-51).

Transplantation of retinal pigment epithelial (RPE) cells into thesubretinal space of RCS rats was found to limit the progress ofphotoreceptor loss and preserve visual function (Li and Turner (1988)Exp Eye Res. 47(6):911-7). In this example, it is demonstrated thatplacenta-derived cells can be used to promote photoreceptor rescue in aRCS model.

Methods & Materials

Cell Transplants.

Cultures of human placental and adult fibroblast cells (passage 10) wereexpanded for 1 passage. All cells were initially seeded at 5,000cells/cm² on gelatin-coated T75 flasks in growth medium ((DMEM:Lowglucose (Invitrogen, Carlsbad, Calif.), 15% (v/v) defined bovine serum(Hyclone, Logan, Utah; Lot#AND18475), 0.001% 2-mercaptoethanol (Sigma,St. Louis, Mo.), 50 Units/milliliter penicillin, 50 microgram/milliliterstreptomycin, 0.25 micrograms per milliliter amphotericin B; Invitrogen,Carlsbad, Calif.)). For subsequent passages, all cells were treated asfollows. After trypsinization, viable cells were counted after trypanBlue staining. Briefly, 50 microliter of cell suspension was combinedwith 50 microliter of 0.04% w/v trypan Blue (Sigma, St. Louis Mo.) andthe viable cell number, was estimated using a heamocytometer. Cells weretrypsinized and washed three times in supplement free-DMEM:Low glucosemedium (Invitrogen, Carlsbad, Calif.). Cultures of human placental andfibroblast cells at passage 11 were trypsinized and washed twice inLeibovitz's L-15 medium (Invitrogen, Carlsbad, Calif.). For thetransplantation procedure, dystrophic RCS rats were anesthetized withxylazine-ketamine (1 milligram/kilogram intraperitoneal (i.p.) of thefollowing mixture: 2.5 milliliters xylazine at 20 milligram/milliliter,5 milliliters ketamine at 100 milligram/milliliter, and 0.5 milliliterdistilled water) and their heads secured by a nose bar. Cells devoid ofserum were resuspended (2×10⁵ cells per injection) in 2 microliter ofLeibovitz, L-15 medium (Invitrogen, Carlsbad, Calif.) and transplantedusing a fine glass pipette (internal diameter 75 to 150 microliter)trans-scerally. Cells were delivered into the dorso-temporal subretinalspace of anesthetized 3 week old dystrophic-pigmented RCS rats (totalN=10/cell type).

Cells were injected unilaterally into the right eye, while the left eyewas injected with carrier medium alone (Sham control; Leibovitz's L-15medium). Viability of residual transplant cells remained at greater than95% as assessed by trypan blue exclusion at the end of the transplantsession. After cell injections were performed, animals were injectedwith dexamethasone (2 milligram/kilogram) for 10 days posttransplantation. For the duration of the study, animals were maintainedon oral cyclosporine A (210 milligram/liter of drinking water; resultingblood concentration: 250-300 microgram/liter) (Bedford Labs, Bedford,Ohio) from 2 days pre-transplantation until end of the study. Food andwater were available ad libitum. Animals were sacrificed at 60 or 90days postoperatively, with some animals being sacrificed at earliertimepoints for histological assessment of short-term changes associatedwith cell transplantation.

ERG Recordings.

Following overnight dark adaptation, animals were prepared for ERGrecording under dim red light, as previously described (Sauve et al.(2004) Vision Res. January; 44(1):9-18). In brief, under anesthesia(with a mixture of 150 milligram/kilogram i.p ketamine, and 10milligram/kilogram i.p. xylazine), the head was secured with astereotaxic head holder and the body temperature monitored through arectal thermometer and maintained at 38° C. using a homeothermicblanket. Pupils were dilated using equal parts of topical 2.5%phenylephrine and 1% tropicamide. Topical anesthesia with 0.75%bupivacaine was used to prevent any corneal reflexes and a drop of 0.9%saline was frequently applied on the cornea to prevent its dehydrationand allow electrical contact with the recording electrode (gold wireloop). A 25-gauge needle inserted under the scalp, between the two eyes,served as the reference electrode. Amplification (at 1-1000 Hz bandpass,without notch filtering), stimulus presentation, and data acquisitionwere provided by the UTAS-3000 system from LKC Technologies(Gaithersburg, Md.). ERGs were recorded at 60 days.

Mixed a- and b-Wave Recording.

For the quantification of dark-adapted b-waves, recordings consisted ofsingle flash presentations (10 microseconds duration), repeated 3 to 5times to verify the response reliability and improve the signal-to-noiseratio, if required. Stimuli were presented at six increasing intensitiesin one log unit steps varying from −3.6 to 1.4 log candila/m² inluminance. To minimize the potential bleaching of rods, inter-stimulusintervals were increased as the stimulus luminance was elevated from 10sec at lowest stimulus intensity to 2 minutes at highest stimulusintensity. The maximum b-wave amplitude was defined as that obtainedfrom the flash intensity series, regardless of the stimulus intensity.The true V_(max) from fitting the data with a Naka-Rushton curve was notused because ERG responses were often erratic at higher luminance levelsin dystrophic animals and showed tendencies for depressed responsesaround 0.4 and 1.4 log candila/m². In order to determine the age atwhich ERG components were obtained or lost, criterion amplitudes wereused: 20 microVolt for a- and b-waves, and 10 microVolt for STR-likeresponses. The amplitude of the b-wave was measured from the a-wavenegative peak up to the b-wave positive apex, and not up to the peak ofoscillations, which can exceed the b-wave apex (Nusinowitz et al. (1999)Invest Ophthalmol Vis Sci. 40(12):2848-58).

Isolation of Rod and Cone Responses.

The double flash protocol was used to determine the isolation of rod andcone responses (Nixon et al. (2001) Clin. Experiment Ophthalmol.29(3):193-6). A probe flash was presented 1 sec after a conditioningflash, using a specific feature of the UTAS-3000 system (LKCTechnologies) with calibrated ganzfeld; assuring complete recharge ofthe stimulator under the conditions used. The role of the conditioningflash in the procedure was to transiently saturate rods so that theywere rendered unresponsive to the probe flash. Response to the probeflash was taken as reflecting cone-driven activity. A rod-driven b-wavewas obtained by subtracting the cone-driven response from the mixedresponse (obtained following presentation of a probe flash alone, i.e.,not preceded by any conditioning flash).

Histology.

Animals were sacrificed with an overdose of urethane (12.5gram/kilogram). The orientation of the eye was maintained by placing a6.0 suture through the superior rectus muscle prior to enucleation.After making a corneal incision, the eyes were fixed with 2.5%parafomaldehyde, 2.5% glutaraldehyde, 0.01% picric acid in 0.1 Mcacodylate buffer (pH 7.4). After fixation, the cornea and lens wereremoved by cutting around the cilliary body. A small nick was made inthe periphery of the dorsal retina prior to removal of the superiorrectus to assist in maintaining orientation. The retinas were thenpost-fixed in 1% osmium tetroxide for 1 hour. After dehydration througha series of alcohols to epoxypropane, the retinas were embedded in TAABembedding resin (TAAB Laboratories, Aldemarston, UK). Semi-thin sectionswere stained with 1% toluidine Blue in 1% borate buffer and the ultrathin sections were contrasted with uranyl acetate and lead citrate.

For Nissl staining, sections were stained with 0.75% cresyl violet(Sigma, St. Louis, Mo.) after which they were dehydrated through gradedalcohols at 70, 95 and 100% twice. They were then placed in xylene(Sigma, St. Louis, Mo.), rinsed with PBS (pH 7.4) (Invitrogen, Carlsbad,Calif.), coverslipped and mounted with DPX mountant (Sigma, St. Louis,Mo.).

Results

ERG Recordings.

At 60 days post-transplant, animals that received placenta-derived cellinjections (n=4) showed no improvement in a-wave (20±20) versus shamcontrols (0), but showed improvement in mixed b-wave (81±72) versus shamcontrols (1.5±2), and good improvement in cone-b-wave (50±19) versussham controls (7±7), and in rod contribution (30%) versus sham controls(0). These results indicated improvement in visual responsiveness whencompared to sham controls. In contrast to transplantation ofplacenta-derived cells, fibroblast transplantations showed noimprovement in any of the parameters tested.

TABLE 28-1 ERG data a-wave mixed b-wave cone b-wave % rod contributionGroup Untreated Treated Untreated Treated Untreated Treated UntreatedTreated Sham 60 days 0 0  7 ± 9 0 23 ± 5  12 ± 16 N/A N/A P (n = 4) 0 20± 20 1.5 ± 2 81 ± 72 7 ± 7 50 ± 19 N/A 30 60 days N.B. Sham = control(medium only), P = Placental cell transplant

Histology.

Following transplantation, there was no histological evidence of aninflammatory reaction and infiltrating immune cells were not observed inNissl-stained sections in the placental cell groups. However, fibroblastimplantations resulted in animal death (n=7) and indications of earlystage inflammatory responses.

REFERENCES

-   Lund et al. (2001) Proc Natl Acad Sci U.S.A. 98(17):9942-7.

Example 29 Chondrogenic Potential of Postpartum-Derived Cells onImplantation in SCID Mice

The chondrogenic potential of cells derived from umbilical cord orplacenta tissue was evaluated following seeding on bioresorbable growthfactor-loaded scaffolds and implantation into SCID mice.

Materials & Methods

Reagents. Dulbecco's Modified Essential Media (DMEM), Penicillin andStreptomycin, were obtained from Invitrogen, Carlsbad, Calif. Fetal calfserum (FCS) was obtained from HyClone (Logan, Utah). Mesenchymal stemcell growth medium (MSCGM) was obtained from Biowhittaker, Walkersville,Md. TGFbeta-3 was obtained from Oncogene research products, San Diego,Calif. GDF-5 was obtained from Biopharm, Heidelberg, Germany(International PCT Publication No. WO96/01316 A1, U.S. Pat. No.5,994,094A). Chondrocyte growth medium comprised DMEM-High glucosesupplemented with 10% fetal calf serum (FCS), 10 milliMolar HEPES, 0.1milliMolar nonessential amino acids, 20 microgram/milliliter L-proline,50 microgram/milliliter ascorbic acid, 100 Unit/milliliter penicillin,100 microgram/milliliter streptomycin, and 0.25 microgram/milliliteramphotericin B. Bovine fibrinogen was obtained from Calbiochem.

Cells.

Human mesenchymal stem cells (hMSC, Lot#2F1656) were obtained fromBiowhittaker, Walkersville, Md. and were cultured in MSCGM according tothe manufacturer's instructions. This lot was tested in the laboratorypreviously in in vitro experiments and was shown to be positive in thechondrogenesis assays. Human adult fibroblasts were obtained fromAmerican Type Culture Collection (ATCC), Manassas, Va. and cultured inGrowth Medium on gelatin-coated tissue culture plastic flasks.Postpartum-derived cells isolated from human umbilical cords(Lot#022703Umb) and placenta (Lot#071003Plac) were prepared aspreviously described (Example 1). Cells were cultured in Growth Mediumon gelatin-coated tissue culture plastic flasks. The cell cultures wereincubated in standard growth conditions. Cells used for experiments wereat passages 5 and 14.

Scaffold.

65/35 Polyglycolic acid (PGA)/Polycaprolactone (PCL) foam scaffolds [4×5centimeters, 1 millimeter thick, Ethylene Oxide (ETO) sterilized]reinforced with Polydioxanone (PDS) mesh (PGA/PCL foam-PDS mesh) wereobtained from Center for Biomaterials and Advanced Technologies (CBAT,Somerville, N.J.). Punches (3.5 millimeters) made from scaffolds wereloaded with either GDF-5 (3.4 micrograms/scaffold), TGFbeta-3 (10nanograms/scaffold), a combination of GDF-5 and TGFbeta-3, or controlmedium, and lyophilized.

Cell Seeding on Scaffolds.

Placenta- and umbilical cord-derived cells were treated with trypsin,and cell number and viability was determined. 0.75×10⁶ cells wereresuspended in 15 microliters of Growth Medium and seeded onto 3.5millimeter scaffold punches in a cell culture dish. The cell-seededscaffold was incubated in a cell culture incubator in standard air with5% CO₂ at 37° C. for 2 hours after which they were placed withincartilage explant rings.

Bovine Cartilage Explants.

Cartilage explants 5 millimeter in diameter were made from cartilageobtained from young bovine shoulder. Punches (3 millimeter) were excisedfrom the center of the explant and replaced with cells seeded 3.5millimeter resorbable scaffold. Scaffolds with cells were retainedwithin the explants using fibrin glue (60 microliter of bovinefibrinogen, 3 milligram/milliliter). Samples were maintained inchondrocyte growth medium overnight, rinsed in Phosphate Buffered Salinethe following day, and implanted into SCID mice.

Animals.

SCID mice ((Mus musculus)/Fox Chase SCID/Male), 5 weeks of age, wereobtained from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.) andCharles River Laboratories (Portage, Mich.). Animals used in the studywere selected without any apparent systematic bias. A tag was placed oneach individual animal cage listing the accession number, implantationtechnique, animal number, species/strain, surgery date, in vivo period,and date of euthanasia. The animals were identified by sequentialnumbers marked on the ear with an indelible ink marker.

Experimental Design.

A total of 42 mice were tested. Two scaffolds were implantedsubcutaneously in each mouse as described below; 42 mice forsubcutaneous implantation; 28 treatments with n-value of 3 pertreatment. The study corresponds to IACUC Approval Number: SkillmanIACUC 01-037. The study lasted six weeks.

SCID Implantation.

A. Body Weights

Each animal was weighed prior to being anesthetized and at necropsy.

B. Anesthesia and Surgical Preparation:

All handling of the SCID mice occurred under a hood. The mice wereindividually weighed and anesthetized with an intraperitoneal injectionof a mixture of KETASET (ketamine hydrochloride [60milligram/kilogram]), ROMPUN (xylazine [10 milligram/kilogram]), andsaline.

After induction of anesthesia, the entire back of the animal from thedorsal cervical area to the dorsal lumbosacral area was clipped free ofhair using electric animal clippers. The area was scrubbed withchlorhexidine diacetate, rinsed with alcohol, dried, and painted with anaqueous iodophor solution of 1% available iodine. Ophthalmic ointmentwas applied to the eyes to prevent drying of the tissue during theanesthetic period. The anesthetized and surgically prepared animal wasplaced in the desired recumbent position.

C. Subcutaneous Implantation Technique:

An approximate 2-cm skin incision was made just lateral to the thoracicspine parallel to the vertebral column. The skin was separated from theunderlying connective tissue via blunt dissection. Each SCID mousereceived 2 treatments that were placed in subcutaneous pockets createdby blunt dissection in each hemithorax through one skin incision.Tacking sutures of 5-0 ETHIBOND EXCEL (polyester) were used to tack theskin to musculature around each scaffold to prevent subcutaneousmigration. Scaffolds were implanted for 6 weeks and then harvested. Theexperimental design is outlined in Table 29-1.

TABLE 29-1 Experimental Design: Treatment (N = 3 per treatment) A. 65/35PGA/PCL Foam + PDS mesh cultured with Placenta-derived cells, EP,TGFbeta3 B. 65/35 PGA/PCL Foam + PDS mesh cultured with Placenta-derivedcells, EP, rhGDF-5 C. 65/35 PGA/PCL Foam + PDS mesh cultured withPlacenta-derived cells, EP, rhGDF-5 + TGFbeta3 D. 65/35 PGA/PCL Foam +PDS mesh cultured with Placenta-derived cells, EP, control E. 65/35PGA/PCL Foam + PDS mesh cultured with Placenta-derived cells, LP,TGFbeta3 F. 65/35 PGA/PCL Foam + PDS mesh cultured with Placenta-derivedcells, LP, rhGDF-5 G. 65/35 PGA/PCL Foam + PDS mesh cultured withPlacenta-derived cells, LP, rhGDF-5 + TGFbeta3 H. 65/35 PGA/PCL Foam +PDS mesh cultured with Placenta-derived cells, LP, control I. 65/35PGA/PCL Foam + PDS mesh cultured with Umbilical cord- derived cells, EP,TGFbeta3 J. 65/35 PGA/PCL Foam + PDS mesh cultured with Umbilical cord-derived cells, EP, rhGDF-5 K. 65/35 PGA/PCL Foam + PDS mesh culturedwith Umbilical cord- derived cells, EP, rhGDF-5 + TGFbeta3 L. 65/35PGA/PCL Foam + PDS mesh cultured with Umbilical cord- derived cells, EP,control M. 65/35 PGA/PCL Foam + PDS mesh cultured with Umbilical cord-derived cells, LP, TGFbeta3 N. 65/35 PGA/PCL Foam + PDS mesh culturedwith Umbilical cord- derived cells, LP, rhGDF-5 O. 65/35 PGA/PCL Foam +PDS mesh cultured with Umbilical cord- derived cells, LP, rhGDF-5 +TGFbeta3 P. 65/35 PGA/PCL Foam + PDS mesh cultured with Umbilical cord-derived cells, LP, control Q. 65/35 PGA/PCL Foam + PDS mesh culturedwith hMSC, TGFbeta3 R. 65/35 PGA/PCL Foam + PDS mesh cultured with hMSC,rhGDF-5 S. 65/35 PGA/PCL Foam + PDS mesh cultured with hMSC, rhGDF-5 +TGFbeta3 T. 65/35 PGA/PCL Foam + PDS mesh cultured with hMSC, control U.65/35 PGA/PCL Foam + PDS mesh cultured with fibroblasts, Adult TGFbeta3V. 65/35 PGA/PCL Foam + PDS mesh cultured with fibroblasts, AdultrhGDF-5 W. 65/35 PGA/PCL Foam + PDS mesh cultured with fibroblasts,Adult rhGDF-5 + TGFbeta3 X. 65/35 PGA/PCL Foam + PDS mesh cultured withfibroblasts, Adult control Y. 65/35 PGA/PCL Foam + PDS mesh, TGFbeta3 Z.65/35 PGA/PCL Foam + PDS mesh, rhGDF-5 AA. 65/35 PGA/PCL Foam + PDSmesh, rhGDF-5 + TGFbeta3 BB. 65/35 PGA/PCL Foam + PDS mesh, control

D. Necropsy and Histologic Preparation

Gross examination was performed on any animals that died during thecourse of the study or were euthanized in moribund condition. Selectedtissues were saved at the discretion of the study director and/orpathologist.

Mice were euthanized by CO₂ inhalation at their designated intervals.Gross observations of the implanted sites were recorded. Samples of thesubcutaneous implantation sites with their overlying skin were excisedand fixed in 10% buffered formalin. Each implant was bisected intohalves, and one half was sent to MPI Research (Mattawan, Mich.) forparaffin embedding, sectioning, and staining with Hematoxylin & Eosin(H&E) and Safranin O (SO).

The data obtained from this study were not statistically analyzed.

Results

New cartilage and bone formation was observed in the majority of thesamples including growth factor-loaded, cell-seeded scaffolds,cell-seeded control scaffolds, and scaffolds loaded with growth factoralone. The extent of new cartilage and bone formation varied within thetreatment and control groups.

Early and Late passage placenta-derived cell seeded scaffolds showed newcartilage and bone formation within the scaffolds. No obviousdifferences in new cartilage and bone formation was observed between thedifferent growth factor-loaded, cell-seeded scaffolds and scaffoldsseeded with cells alone. Compared to control scaffolds (without growthfactors and without cells), it appeared that there was greater extent ofnew cartilage formation in cell-seeded scaffolds both with and withoutgrowth factors and in growth factor-loaded scaffolds alone. Newcartilage formation with placenta-derived cell-seeded scaffolds wassimilar to MSC- and fibroblast-seeded scaffolds.

In growth factor-treated and control scaffolds seeded with umbilicalcord-derived cells at early and late passage, new cartilage and boneformation were observed. The extent of cartilage formation appeared tobe less than that seen with placenta-derived cells. No one sample showedextensive cartilage formation as seen with the placenta-derived cells.Bone formation appeared to be higher in scaffolds seeded with umbilicalcord-derived cells on scaffolds containing both TGFbeta-3 and rhGDF-5.

hMSC-loaded scaffolds also showed new cartilage and bone formation. Theextent of new cartilage and bone formation was similar for all the hMSCtreatment groups. Human adult fibroblast seeded scaffolds alsodemonstrated new cartilage and bone formation. Results were similar tothose obtained with placenta-derived cells and hMSCs

In the control group, in which growth factor-loaded scaffolds orscaffold alone were placed in cartilage rings and implanted, newcartilage and bone formation were also observed. Not surprisingly, theextent of new cartilage formation was greater in scaffolds with growthfactor than in scaffolds without growth factor. Increased bone formationwas present in the control with the combination of the two tested growthfactors.

New cartilage formation was observed adjacent to the cartilage explantrings as well as within the scaffolds. New cartilage formation withinthe scaffolds adjacent to the cartilage rings could be a result ofchondrocyte migration. Cartilage formation seen as islands within thescaffolds may be a result of either migration of chondrocytes within thescaffolds, differentiation of seeded cells or differentiation ofendogenous mouse progenitor cells. This observation stems from the factthat in control growth factor-loaded scaffolds with no seeded cells,islands of chondrogenic differentiation were observed. New boneformation was observed within the scaffolds independently and alsoassociated with chondrocytes. Bone formation may have arisen fromosteoblast differentiation as well as endochondral ossification.

It is difficult to separate new cartilage and bone formation associatedwith chondrocytes that migrated versus that from any chondrogenic andosteogenic differentiation of seeded cells that may have occurred.Staining of sections with specific human antibodies may distinguish thecontribution of the seeded cells to the observed chondrogenesis andosteogenesis.

It is also possible that placenta-derived cells and umbilicalcord-derived cells stimulated chondrocyte migration.

Abundant new blood vessels were observed with the scaffolds loaded withplacenta-derived cells and umbilical cord-derived cells. Blood vesselswere abundant in areas of bone formation. New blood vessels were alsoobserved within the hMSC- and fibroblast-seeded scaffolds associatedwith new bone formation.

Systemic effects of the adjacent scaffold (with growth factor (GF)) onthe control scaffolds (no GF, no cells) on promoting new cartilage andbone formation cannot be ruled out. Analysis of new cartilage and boneformation in scaffolds, taking into consideration the scaffoldsimplanted adjacent to it in SCID mice, showed no clear pattern ofsystemic effect of growth factor from the adjacent scaffold.

Summary.

Results showed that new cartilage and bone formation were observed ingrowth factor and control scaffolds seeded with placenta- and umbilicalcord-derived cells. Results with placenta-derived cells were similar tothat seen with human mesenchymal stem cells, while the extent of newcartilage like tissue formation was slightly less pronounced inumbilical cord-derived cells. Growth factor-loaded scaffolds implantedwithout cells also demonstrated new cartilage and bone formation. Thesedata indicate that new cartilage formation within the scaffolds mayarise from chondrocytes that migrated from the bovine explants, fromchondrogenic differentiation of endogenous progenitor cells, and fromchondrogenic differentiation of seeded cells.

These results suggest that placenta- and umbilical cord-derived cellsundergo chondrogenic and osteogenic differentiation. These results alsosuggest that placenta- and umbilical cord-derived cells may promotemigration of chondrocytes from the cartilage explant into the scaffolds.Abundant new blood vessels were also observed in the scaffoldsespecially associated with new bone formation.

Example 30 Use of Postpartum-Derived Cells in Nerve Repair

Retinal ganglion cell (RGC) lesions have been extensively used as modelsfor various repair strategies in the adult mammalian CNS. It has beendemonstrated that retrobulbar section of adult rodent RGC axons resultsin abortive sprouting (Zeng et al., 1995) and progressive death of theparent cell population (Villegas-Perez et al., 1993). Numerous studieshave demonstrated the stimulatory effects of various exogenous andendogenous factors on the survival of axotomized RGC's and regenerationof their axons (Yip and So, 2000; Fischer et al., 2001). Furthermore,other studies have demonstrated that cell transplants can be used topromote regeneration of severed nerve axons (Li et al., 2003;Ramon-Cueto et al., 2000). Thus, these and other studies havedemonstrated that cell based therapy can be utilized for the treatmentof neural disorders that affect the spinal cord, peripheral nerves,pudendal nerves, optic nerves or other diseases/trauma due to injury inwhich nervous damage can occur.

Self-assembling peptides (PuraMatrix™, U.S. Pat. Nos. 5,670,483,5,955,343, U.S. Published Application No. 2002/0160471, InternationalPatent Publication No. WO 02/062969) have been developed to act as ascaffold for cell-attachment to encapsulate cells in 3-D, plate cells in2-D coatings, or as microcarriers in suspension cultures.Three-dimensional cell culture has required either animal-derivedmaterials (mouse sarcoma extract), with their inherent reproducibilityand cell signaling issues, or much larger synthetic scaffolds, whichfail to approximate the physical nanometer-scale and chemical attributesof native ECM. RAD 16 (NH₂-RADARADARADA-COOH) and KLD(NH₂-KLDLKLDLKLDL-COOH) are synthesized in small (RAD16 is 5 nanometers)oligopeptide fragments that self-assemble into nanofibers on a scalesimilar to the in vivo extracellular matrix (ECM) (3D Matrix, IncCambridge, Mass.). The self-assembly is initiated by mono- or di-valentcations found in culture media or the physiological environment. In theprotocols described in this example, RAD 16 was used as a microcarrierfor the implantation of postpartum cells into the ocular defect. In thisexample, it is demonstrated that transplants of postpartum-derived cellsPPDCs) can provide efficacy in an adult rat optic nerve axonalregeneration model.

Methods & Materials

Cells.

Cultures of human adult PPDCs (umbilical cord and placenta) andfibroblast cells (passage 10) were expanded for 1 passage. All cellswere initially seeded at 5,000 cells/cm² on gelatin-coated T75 flasks inGrowth Medium ((DMEM:Low glucose (Invitrogen, Carlsbad, Calif.), 15%(v/v) defined bovine serum (Hyclone, Logan, Utah; Lot#AND18475), 0.001%2-mercaptoethanol (Sigma, St. Louis, Mo.), 100 Units per milliliterpenicillin, 100 micrograms per milliliter streptomycin, 0.25 microgramsper milliliter amphotericin B; Invitrogen, Carlsbad, Calif.). At passage11 cells were trypsinized and viability was determined using trypan bluestaining. Briefly, 50 microlitres of cell suspension was combined with50 microlitres of 0.04% w/v trypan blue (Sigma, St. Louis Mo.) and theviable cell number, was estimated using a hemocytometer. Cells were thenwashed three times in supplement free-Leibovitz's L-15 medium(Invitrogen, Carlsbad, Calif.). Cells were then suspended at aconcentration of 200,000 cells in 25 microliters of RAD-16 (3DM Inc.,Cambridge, Mass.) which was buffered and made isotonic as permanufacturer's recommendations. One hundred microliters of supplementfree Leibovitz's L-15 medium was added above the cell/matrix suspensionto keep it wet till use. These cell/matrix cultures were maintainedunder standard atmospheric conditions until transplantation occurred. Atthe point of transplantation the excess medium was removed.

Animals and Surgery.

Long Evans female rats (220-240 gram body weight) were used. Underintraperitoneal tribromoethanol anesthesia (20 milligram/100 grams bodyweight), the optic nerve was exposed, and the optic sheath was incisedintraorbitally at approximately 2 millimeter from the optic disc, thenerve was lifted from the sheath to allow complete transsection withfine scissors (Li et al., 2003). The completeness of transection wasconfirmed by visually observing complete separation of the proximal anddistal stumps. The control group consisted of lesioned rats withouttransplants. In transplant rats cultured postpartum cells seeded inRAD-16 were inserted between the proximal and distal stumps using a pairof microforceps. Approximately 75,000 cells in RAD-16 were implantedinto the severed optic nerve. Cell/matrix was smeared into the severedcut using a pair of fine microforceps. The severed optic nerve sheathwas closed with 10/0 black monofilament nylon (ETHICON, Edinburgh, UK).Thus, the gap was closed by drawing the cut proximal and distal ends ofthe nerve in proximity with each other.

After cell injections were performed, animals were injected withdexamethasone (2 milligrams/kilogram) for 10 days post transplantation.For the duration of the study, animals were maintained on oralcyclosporine A (210 milligrams/liter of drinking water; resulting bloodconcentration: 250-300 micrograms/liter) (Bedford Labs, Bedford, Ohio)from 2 days pre-transplantation until end of the study. Food and waterwere available ad libitum. Animals were sacrificed at either 30 or 60days posttransplantation.

CTB Application.

Three days before animals were sacrificed, under anesthesia, a glassmicropipette with a 30-50 millimeter tip was inserted tangentiallythrough the sclera behind the lens, and two 4-5 microliter aliquots of a1% retrograde tracer-cholera toxin B (CTB) aqueous solution (ListBiologic, Campbell, Calif.) was injected into the vitreous. Animals wereperfused with fixative and optic nerves were collected in the samefixative for 1 hour. The optic nerves were transferred into sucroseovernight. Twenty micrometer cryostat sections were incubated in 0.1molar glycine for 30 minutes and blocked in a PBS solution containing2.5% bovine serum albumin (BSA) (Boeringer Mannheim, Mannheim, Germany)and 0.5% triton X-100 (Sigma, St. Louis, Mo.), followed by a solutioncontaining goat anti-CTB antibody (List Biologic, Campbell, Calif.)diluted 1:4000 in a PBS containing 2% normal rabbit serum (NRS)(Invitrogen, Carlsbad, Calif.), 2.5% BSA, and 2% Triton X-100 (Sigma,St. Louis, Mo.) in PBS, and incubated in biotinylated rabbit anti-goatIgG antibody (Vector Laboratories, Burlinghame, Calif.) diluted 1:200 in2% Triton-X100 in PBS for 2 hours at room temperature. This was followedby staining in 1:200 streptavadin-green (Alexa Flour 438; MolecularProbes, Eugene, Oreg.) in PBS for 2 hours at room temperature. Stainedsections were then washed in PBS and counterstained with propidiumiodide for confocal microscopy.

Histology Preparation.

Briefly, 5 days after CTB injection, rats were perfused with 4%paraformaldehyde. Rats were given 4 cubic centimeters of urethane andwere then perfused with PBS (0.1 molar) then with 4% Paraformaldehyde.The spinal cord was cut and the bone removed from the head to expose thecolliculus. The colliculus was then removed and placed in 4%Paraformaldehyde. The eye was removed by cutting around the outside ofthe eye and going as far back as possible. Care was given not to cut theoptic nerve that lies on the underside of the eye. The eye was removedand the muscles were cut exposing the optic nerve this was then placedin 4% Paraformaldehyde.

Results

Lesions Alone.

One month after retrotubular section of the optic nerve, a number ofCTB-labeled axons were identified in the nerve segment attached to theretina. In the 200 micrometers nearest the cut, axons were seen to emita number of collaterals at right angles to the main axis and terminateas a neuromatous tangle at the cut surface. In this cut between theproximal and distal stumps, the gap was observed to be progressivelybridged by a 2-3 millimeter segment of vascularized connective tissue;however, no axons were seen to advance into this bridged area. Thus, inanimals that received lesion alone no axonal growth was observed toreach the distal stump.

RAD-16 Transplantation.

Following transplantation of RAD-16 into the cut, visible ingrowth ofvascularized connective tissue was observed. However, no axonal ingrowth was observed between the proximal and distal stumps. The resultsdemonstrate that application of RAD-16 alone is not sufficient forinducing axonal regeneration in this situation.

Transplantation of Postpartum-Derived Cells.

Transplantation of postpartum-derived cells into the severed optic nervestimulated optic nerve regrowth. Some regrowth was also observed inconditions in which fibroblast cells were implanted, although this wasminimal as compared with the regrowth observed with the transplantedplacenta-derived cells. Optic nerve regrowth was observed in 4/5 animalstransplanted with placenta-derived cells, 3/6 animals transplanted withadult dermal fibroblasts, and in 1/4 animals transplanted with umbilicalcord-derived cells. In situations where regrowth was observed, CTBlabeling confirmed regeneration of retinal ganglion cell axons, whichwere demonstrated to penetrate through the transplant area. GFAPlabeling was also performed to determine the level of glial scarring.The GFAP expression was intensified at the proximal stump with someimmunostaining being observed through the reinervated graft.

Summary.

These results demonstrate that transplanted human adultpostpartum-derived cells are able to stimulate and guide regeneration ofcut retinal ganglion cell axons.

REFERENCES

-   1) Zeng B Y, Anderson P N, Campbell G, Lieberman A R. 1995. J. Anat.    186:495-508.-   2) Villegas-Perez M P, Vidal-Sanz M, Bray G M, Aguayo A J. 1988. J    Neurosci. 8:265-80.-   3) Yip H K, So K F. 2000. Prog Retin Eye Res. 19: 559-75.-   4) Fischer D, Heiduschka P, Thanos S. 2001. Exp Neurol. 172: 257-72.-   5) Ramon-Cueto A, Cordero M I, Santos-Benito F F, Avila J. 2000.    Neuron 25: 425-35.

While the present invention has been particularly shown and describedwith reference to the presently preferred embodiments, it is understoodthat the invention is not limited to the embodiments specificallydisclosed and exemplified herein. Numerous changes and modifications maybe made to the preferred embodiment of the invention, and such changesand modifications may be made without departing from the scope andspirit of the invention as set forth in the appended claims.

What is claimed:
 1. A method of producing a population ofplacenta-derived cells by expanding an isolated human placenta-derivedcell in culture and obtaining a homogenous population ofplacenta-derived cells, wherein said isolated human placenta-derivedcell is obtained from human postpartum placenta substantially free ofblood, and wherein said cell population self-renews and expands inculture, is multipotent, and has the following characteristics: a)produces CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2, HLA-A,B,C,and granulocyte chemotactic protein-2 (GCP-2); b) does not produce CD31,CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G or HLA-DR-DP,DQ; c) does not produce GRO-alpha and oxidized low density lipoproteinreceptor, as detected by flow cytometry; d) expresses, relative to ahuman fibroblast, mesenchymal stem cell, or iliac crest bone marrowcell, increased levels of oxidized low density lipoprotein receptor 1mRNA and renin; and e) has the ability to undergo at least 40 populationdoublings in culture.
 2. The method of claim 1, wherein said expandingsaid isolated placenta-derived cell comprises culturing in RPMI1640medium, Ham's F10 medium, Ham's F12 medium, Mesenchymal Stem Cell GrowthMedia, Iscove's modified Dulbecco's medium, Dulbecco's Modified Eagle'sMedium (DMEM)-high glucose, DMEM-low glucose, advanced DMEM,DMEM/MCDB201, CELL-GRO FREE, DMEM/F12, or Eagle's basal medium.
 3. Themethod of claim 1, wherein said expanding comprises culturing saidplacenta-derived cell in medium comprising DMEM-low glucose, serum,beta-mercaptoethanol, and an antibiotic agent.
 4. The method of claim 1,wherein the culture medium comprises serum.
 5. The method of claim 1,wherein the culture medium is protein-free culture medium.
 6. The methodof claim 1, wherein the culture medium is serum-free culture medium. 7.The method of claim 1, wherein the cell population further has thefollowing characteristics: a) secretion of monocyte chemotactic protein1 (MCP-1), interleukin-6 (IL-6), interleukin 8 (IL8), hepatocyte growthfactor (HGF), keratinocyte growth factor (KGF), heparin-bindingepidermal growth factor (HB-EGF), brain-derived neurotrophic factor(BDNF), tissue inhibitor of matrix metalloproteinase 1 (TIMP1),thrombopoietin (TPO), macrophage inflammatory protein 1alpha (MIP1a),Rantes (regulated on activation, normal T cell expressed and secreted),thymus and activation-regulated chemokine (TARC), and Eotaxin; and b)lack of secretion of fibroblast growth factor (FGF), vascularendothelial growth factor (VEGF), angiopoietin 2 (ANG2), plateletderived growth factor (PDGF-bb), transforming growth factor beta2(TGFbeta2), macrophage inflammatory protein 1beta (MIP1b), 1309, andmacrophage-derived chemokine (MDC), as detected by ELISA.
 8. The methodof claim 1, wherein the isolated human placenta-derived cell is of aneonatal origin.
 9. The method of claim 1, wherein the isolated humanplacenta-derived cell is of a maternal origin.
 10. The method of claim1, wherein the cell population expands in the following culture media inorder of growth: Growth medium >Mesenchymal Stem Cell Growth Medium(MSCGM)>Iscove's+10% serum=DMEM-high glucose+10% serum=Ham's F12+10%serum=RPMI1640+10% serum.
 11. The method of claim 1, wherein the cellpopulation has the ability to differentiate into a mesodermal,ectodermal, or endodermal phenotype.
 12. The method of claim 1, whereinsaid cell isolated from human postpartum placenta substantially free ofblood is produced by contacting said human postpartum placentasubstantially free of blood with a composition comprising a matrixmetalloprotease, a mucolytic enzyme that digests hyaluronic acid, and aneutral protease, and obtaining the cells yielded by said contacting.13. The method of claim 1, wherein the isolated homogeneous cellpopulation is identified by ATCC Accession No. PTA-6074.
 14. The methodof claim 1, wherein the isolated homogeneous cell population isidentified by ATCC Accession No. PTA-6075.
 15. The method of claim 1,wherein the isolated homogeneous cell population is identified by ATCCAccession No. PTA-6079.